Combination method for treatment of cancer

ABSTRACT

The invention relates to methods of treating tumours comprising delivering an oncolytic virus or oncolytic viral RNA via direct injection or systemic administration or intravesicular administration to the tumour or cancer in combination with the co-administration of an immuno-stimulatory agent via the systemic route to a mammal.

FIELD

The present invention relates to methods of treating tumours comprisingdelivering an oncolytic virus or oncolytic viral RNA via directinjection or systemic administration to the tumour or cancer incombination with the co-administration of an immuno-stimulatory agentvia the systemic route to a mammal.

RELATED APPLICATIONS

This application claims the benefit of priority to AustralianProvisional Patent Application No. 2014900647 entitled “Combinationmethod for treatment of cancer” dated 27 Feb. 2014, the entire contentsof which are incorporated herein by reference.

BACKGROUND

Several new immunotherapeutic approaches for melanoma treatment haveshown promising results in recent clinical trials. The antibody basedtherapy, ipilimumab was approved by the U.S. Food and DrugAdministration (FDA) in March 2011, having demonstrated improved overallsurvival in patients with metastatic melanoma in Phase III clinicaltrials. Ipilimumab (Trade name: Yervoy®, Bristol-Myers Squibb) is amonoclonal antibody that targets the protein cytotoxic Tlymphocyte-associated antigen 4 (CTLA-4)(CD152). Signaling throughCTLA-4 is known to have an inhibitory effect on cytotoxic T-lymphocytes(CTLs) and by blocking this inhibitory signal with ipilimumab, CTLs areable to successfully target the destruction of cancer cells. In melanomapatients, the overall survival with ipilimumab alone was 10.1 monthscompared to 6.4 months for patients treated with a glycoprotein 100(gp100) peptide vaccine [Hodi, et al., 2010]. In phase III studies, theresponse rate of previously treated advanced melanoma patients was 11%following ipilimumab treatment alone, and 15% in ipilimumab plusdacarbazine therapy of treatment-naïve patients [Hodi, et al., 2010;Robert, et al., 2011]. In both of these trials, improved overallsurvival with ipilimumab was clearly shown, as evident by reductions inthe probability of death by 34% (compared with vaccine) and 28%(compared with dacarbazine alone). When ipilimumab was used incombination with dacarbazine, the median duration of best overallresponse was 19.3 months compared with 8.1 months with dacarbazinemonotherapy.

Another immune checkpoint molecule being targeted for the immunotherapyof cancer, is programmed cell death 1 (PD-1)(CD279) found on the surfaceof activated T cells, B cells and myeloid cells [Keir, et al., 2008].When the molecule programmed cell death 1 ligand 1 (PD-L1)(CD274) orPDL-2 (CD273) binds to the PD-1 receptor, the T cell is inhibitedthereby limiting potential anti-tumoural responses. Currently there area range of antibodies designed to target PD-1 and PD-L1 as a means ofstimulating anti-tumoural T cell immunity, namely nivolumab (BMS-936558,Bristol-Myers Squibb) and the anti-PD-L1 antibody BMS-936559 [Brahmer,et al., 2012; Topalian, et al., 2012]. These antibodies against PD-1 andPD-L1 have been used in a range of preclinical studies in combinationwith CTLA-4 and in Phase III human trials [Curran et al, 2010; Merelli,2014; Ott et al, 2013].

Coxsackievirus A21 (CVA21) is a naturally occurring picornavirus thathas the capacity to preferentially infect and destroy malignant cellsbearing the virus-cell entry receptor intercellular adhesion molecule-1(ICAM-1) and/or decay accelerating factor (DAF). Previously we havedemonstrated the efficacy of CVA21 against melanoma cells in a range ofpre-clinical xenograft models using immune-deficient mice, as well asagainst other cancers such as breast cancer, prostate cancer, colorectalcancer and ovarian cancer [Shafren, et al., 2004; Au, et al., 2005; Auet al., 2007; WO2001/037866]; and against hematologic cancers[WO/2006/017914].

There remains a need for new and improved methods for the treatment,alleviation, or prevention of cancer and for methods of improvingsurvival in subjects with tumors or cancer.

SUMMARY OF INVENTION

The inventors propose that CVA21 infection and subsequent lysis oftumour cells, results in the release of cellular debris that containsmelanoma antigens that stimulate anti-tumoural immunity, thereby actingas a personalised in situ cancer vaccine. First we established animmune-competent B16-ICAM-1 melanoma model where B16 cells, normallyresistant to CVA21, were transfected with human ICAM-1 to make thempermissive to infection. Given the generally poor induction ofanti-tumoural immunity of most cancer vaccines [Blanchard, et al., 2013;Garbe, et al., 2011], we investigated the use of the immunostimulatoryanti-PD-1 antibody in combination with CVA21 virotherapy and separatelythe use of the immune-stimulatory anti-CTLA-4 antibody in combinationwith CVA21 virotherapy, as well as the use of CVA21 virotherapy incombination with both anti-PD-1 antibody and anti-CTLA-4 antibody.

As demonstrated herein the use of immunostimulatory agents targetingimmune checkpoint molecules, exemplified herein by the use of a murineanti-PD-1 antibody and a murine anti-CTLA-4 antibody, in combinationwith oncolytic CVA21 would be therapeutically beneficial in reducingtumour burden, rates of tumour ulceration and prolong survival in amurine B16-ICAM-1 model of melanoma.

In one aspect the invention provides a method for the treatment ofcancer in a subject, the method comprising delivering an oncolytic virusor oncolytic viral RNA via direct injection to a tumor or systemicadministration to the subject in combination with the co-administrationof an immuno-stimulatory agent via the systemic route to the subject.

In one aspect the invention provides a method for the treatment ofbladder cancer in a subject, the method comprising delivering anoncolytic virus or oncolytic viral RNA via direct injection to a tumoror systemic administration or intravesicular administration to thesubject in combination with the co-administration of animmuno-stimulatory agent via the systemic route to the subject.

In an embodiment the oncolytic virus or oncolytic viral RNA is selectedfrom the group consisting of Family Picornaviridae. In an embodiment theoncolytic virus or oncolytic viral RNA selected from the groupconsisting of Family Picornaviridae virus that bind to intercellularadhesion molecule-1 (ICAM-1) and/or decay-accelerating factor (DAF) onthe surface of the tumour cell. In an embodiment the oncolytic virus oroncolytic viral RNA is selected from the group consisting of genusenterovirus that bind to intercellular adhesion molecule-1 (ICAM-1)and/or decay-accelerating factor (DAF) on the surface of the tumourcell. In an embodiment the enterovirus is a human enterovirus C. In anembodiment the oncolytic virus or oncolytic viral RNA is selected fromthe group consisting of Group A Coxsackievirus that bind tointercellular adhesion molecule-1 (ICAM-1) and/or decay-acceleratingfactor (DAF) on the surface of the tumour cell. In an embodiment theoncolytic virus or oncolytic viral RNA is Coxsackievirus A21.

In an embodiment the immuno-stimulatory agent is an agent that targetsan immune checkpoint molecule selected from the group consisting ofPD-1, PD-L1, PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86,B7-H3, B7-H4, B7RP1, ICOS, TIM3, GAL9, CD28 or OX-40. In an embodimentthe immunostimulatory agent is selected from the group consisting of anagent that specifically binds to the surface expressed PD-1, PD-L1,PD-L2, CTLA-4 or OX-40. In an embodiment the immunostimulatory agent isselected from the group consisting of monoclonal antibodies thatspecifically binds to the surface expressed PD-1, PD-L1, PD-L2, CTLA-4or OX-40.

In an embodiment the method comprises delivering the oncolytic virus oroncolytic viral RNA via direct injection or systemic administration orintravesicular administration prior to the administration of animmuno-stimulatory agent via the systemic route.

In an embodiment the method comprises delivering the oncolytic virus oroncolytic viral RNA via direct injection or systemic administration orintravesicular administration following the administration of animmuno-stimulatory agent via the systemic route to the subject.

In an embodiment the treatment provides increased survival time for asubject compared to estimated survival time in the absence of saidtreatment. In an embodiment the treatment provides retardation of tumourgrowth compared to estimated tumour growth in the absence of saidtreatment.

In one aspect the invention provides use of oncolytic virus or oncolyticviral RNA for the manufacture of a medicament for the treatment of asubject having cancer, wherein said medicament is for use in combinationwith an immuno-stimulatory agent delivered via the systemic route to thesubject, and wherein said medicament is for delivery via directinjection to the tumor or systemic administration to the subject.

In one aspect the invention provides use of oncolytic virus or oncolyticviral RNA for the manufacture of a medicament for the treatment of asubject having bladder cancer, wherein said medicament is for use incombination with an immuno-stimulatory agent delivered via the systemicroute to the subject, and wherein said medicament is for delivery viadirect injection to a tumor or systemic administration or intravesicularadministration to the subject.

In an embodiment of said use, the oncolytic virus or oncolytic viral RNAis for administration to the tumour via direct injection or systemic orintravesicular administration prior to the administration of animmuno-stimulatory agent via the systemic route to the mammal. In anembodiment of said use, the medicament comprising an oncolytic virus oroncolytic viral RNA is for administration to the tumour via directinjection or systemic or intravesicular administration following theadministration of an immuno-stimulatory agent via the systemic route tothe mammal.

In one aspect the invention provides an oncolytic virus or oncolyticviral RNA for use in treatment of a subject having cancer, wherein saiduse is in combination with an immuno-stimulatory agent, wherein in saiduse the oncolytic virus or oncolytic viral RNA is administered to saidsubject via direct injection to a tumor or systemic administration tothe subject and said immuno-stimulatory agent is administered via thesystemic route to the subject.

In one aspect the invention provides an oncolytic virus or oncolyticviral RNA for use in treatment of a subject having bladder cancer,wherein said use is in combination with an immuno-stimulatory agent,wherein in said use the oncolytic virus or oncolytic viral RNA isadministered to said subject via direct injection to a tumor or systemicadministration or intravesicular administration to the subject and saidimmuno-stimulatory agent is administered via the systemic route to thesubject.

In an embodiment the administration of said oncolytic virus or oncolyticviral RNA is prior to the administration of the immuno-stimulatoryagent. In an embodiment the administration of said oncolytic virus oroncolytic viral RNA is following the administration of theimmuno-stimulatory agent.

In an embodiment the mammal or subject is a human.

In an embodiment the cancer or tumour is selected from the groupconsisting of prostate cancer, breast cancer, ovarian cancer, lymphoidcancer, leukemia, brain cancer, lung cancer, colorectal cancer, thyroidcancer, renal cancer, adrenal cancer, liver cancer, stomach cancer,intestinal cancer, bladder cancer, cancer of the kidney, multiplemyeloma, non-small cell lung cancer (NSCLC), pancreatic cancer,glioblastoma and melanoma.

In an embodiment the cancer or tumour is melanoma.

In an embodiment the cancer or tumor is metastatic cancer or ametastatic tumor.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: Overview of immune-competent murine melanoma protocol. A) Flowdiagram showing treatment groups. Animals were first treated with eitherCVA21 (1×10⁸ TCID 50 [5×10⁹ TCID 50/kg]) intratumourally, UV-inactivatedCVA21 or saline, followed by intraperitoneal injections with the murineisotype control antibody or the anti-PD-1 antibody (12.5 mg/kg). B) Timeline showing the schedule of treatments (green triangles and orangestars) and monitoring procedures. The experiment was terminated on day45. (NTC=No Tumour Control).

FIG. 2: Average body weights of mice (g) vs time (days). Error barsindicate standard error. The dotted lines at day 12, 15, 18 and 21 showeach cycle of therapy. Using multiple t-tests corrected for multiplecomparisons (Holm-Sidak method), there were no statistically significantdifferences between the average body weights of treatment groups and theno tumour control (NTC) mice, except for the UV-CVA21+anti-PD-1 group atday 31. Some fluctuations in mean weights were observed due to theeuthanasia of mice over the duration of the study.

FIG. 3: Individual body weights of mice (g) vs time (days). The dottedlines indicate the four rounds of therapy at day 12, 15, 18 and 21. A)No Tumour control group, B) Saline+Control Ab, C) Saline+anti-PD-1, D)UV-CVA21+Control Ab, E) UV-CVA21+anti-PD-1, F) CVA21+Control Ab, G)CVA21+anti-PD-1.

FIG. 4: Individual tumour volumes from each mouse following combinationimmunotherapy and CVA21 virotherapy on B16-ICAM-1 murine melanomatumours (n=8 per group). C57BL/6 mice were injected with B16-ICAM-1cells intradermally on the hind flank. On days 12, 15, 18 and 21 tumourswere injected intratumourally with either saline, CVA21 (1×10⁸ TCID₅₀[5×10⁹ TCID₅₀/kg]) or UV-inactivated CVA21 (1×10⁸ TCID₅₀ [5×10⁹TCID₅₀/kg]), in combination with the control or anti-PD-1 antibody (12.5mg/kg respectively). Treatment days are indicated by the dotted lines.During the treatment period there was a noticeable trend of delayedtumour growth in the anti-PD-1 treated groups vs control antibodygroups. The tumour take rate in the saline+anti-PD-1 treatment group wasquite variable and at least three animals were euthanased before thecourse of therapy was completed.

FIG. 5: Detection of A) viremia and B) anti-CVA21 neutralisingantibodies following CVA21 oncolytic virotherapy in combination withanti-PD-1 immunotherapy. Graphs show mean+SEM (n=8 at the commencementof the study). The differences in neutralising antibody levels betweenthe CVA21+anti-PD-1 antibody vs CVA21+control antibody were significanton day 33=0.04 one-tailed t-test).

FIG. 6: Survival of C57BL/6 mice following treatment saline, UV-CVA21 orCVA21 in combination with the control antibody or anti-PD-1. The dottedline at day 45 indicates the end/termination of the experiment.

FIG. 7: Survival of C57BL/6 mice following treatment with either saline,UV-inactivated CVA21, CVA21 in combination with the control antibody oranti-PD-1 antibody. Survival curve comparisons between A) Saline+controlAb vs CVA21+control Ab, B) Saline+anti-PD-1 vs CVA21+anti-PD-1, C)Saline+control Ab vs Saline+anti-PD-1, D) CVA21+control Ab vsCVA21+anti-PD-1, E) Saline+control Ab vs CVA21+anti-PD-1, F)UV-CVA21+anti-PD-1 vs CVA21+anti-PD-1 The dotted lines show the 50%survival cut offs and corresponding median survival time.

FIG. 8: Overview of immune-competent murine melanoma protocol, asdescribed in Example 2. Time line showing the schedule of treatments(blue circles [anti-PD-1 antibody], orange squares [CVA21]) andmonitoring procedures (red triangles [blood collection] and greentriangles [tumor measurements and body weight measurements]). Animalswere first treated with either saline or CVA21 (1×10⁸ TCID₅₀/injection[5.56×10⁹ TCID₅₀/kg]), followed by intraperitoneal injections with themurine isotype control antibody or the anti-PD-1 antibody (12.5 mg/kg).The experiment was terminated on day 66.

FIG. 9: Individual tumor volumes from each mouse following combinationimmunotherapy and CVA21 virotherapy on B16-ICAM-1 murine melanoma tumors(n=12 per group). A) Saline+Control Ab, B) Saline+anti-PD-1, C)CVA21+Control Ab, D) CVA21+anti-PD-1. C57BL/6 mice were injected withB16-ICAM-1 cells intradermally on the hind flank. On days 6, 9, 12 and15 tumors were injected intratumorally with either saline or CVA21(1×10⁸ TCID₅₀ [5:56×10⁹ TCID₅₀/kg]), in combination with the control oranti-PD-1 antibody (12.5 mg/kg respectively). Treatment days areindicated by the dotted lines. Additional intratumoral virus injections(1×10⁸ TCID₅₀ [5:56×10⁹ TCID₅₀/kg) were administered at days 19, 26, 33and 40. During the treatment period there was a noticeable trend ofdelayed tumor growth in the anti-PD-1 treated groups vs control antibodygroups.

FIG. 10: Tumor volumes of secondary B16 tumor nodules. Individual tumorvolumes from each mouse following rechallenge with B16 tumor cells(1×10⁵) on the right hind flank at day 31. A) Saline+Control Ab, B)Saline+anti-PD-1, C) CVA21+Control Ab, D) CVA21+anti-PD-1. All animalseventually developed palpable tumors, however, there was a trendindicating that the onset of B16 tumor growth was delayed byCVA21+anti-PD-1 therapy.

FIG. 11: Survival of C57BL/6 mice following treatment with saline orCVA21 in combination with the control antibody or anti-PD-1. A) When thesaline+anti-PD-1 survival curve was compared with the CVA21+anti-PD-1treatment group, there was a statistical difference (p=0.0026 Log-rank[Mantel-Cox] test). B) CVA21 used in combination with the anti-PD-1antibody gave a significant survival advantage (median survival of 45 vs60 days for saline+anti-PD-1 and CVA21+anti-PD-1 respectively). Thestudy was terminated at day 66.

FIG. 12: Overview of immune-competent murine melanoma protocol. Timeline showing the schedule of treatments (blue circles [anti-CTLA-4antibody] and orange squares [CVA21]) and monitoring procedures (redtriangles [blood collection] and green triangles [tumor measurements andbody weight measurements]). The experiment was terminated on day 77.

FIG. 13: Tumor volumes following combination immunotherapy and CVA21virotherapy on B16-ICAM-1 murine melanoma tumors. (A) Average tumorvolumes (mm3)±S.E.M. following treatment with either saline+controlantibody, saline+anti-CTLA-4, CVA21+control antibody orCVA21+anti-CTLA-4. (B & C) Individual tumor volumes from each mousefollowing combination immunotherapy and CVA21 virotherapy on B16-ICAM-1murine melanoma tumors. C57BL/6 mice were injected with B16-ICAM-1 cellsintradermally on the hind flank. On days 7, 10, 13 and 16 tumors wereinjected intratumorally with either saline or CVA21 (1×10⁸ TCID₅₀[5.56×10⁹ TCID₅₀/kg]), in combination with the control or anti-CTLA-4antibody (12.5 mg/kg respectively). Treatment days are indicated by thedotted lines.

FIG. 14: Tumor volumes of secondary B16 tumor nodules. (A) Percentageincidence of secondary tumor development in saline+control antibody,saline+anti-CTLA-4, CVA21+control antibody, and CVA21+anti-CTLA-4treated mice at the conclusion of the study (day 77). (B) Graph of tumorincidence vs days. Mice were rechallenged with B16 tumor cells on thehind flank at day 37 and tumors monitored up until day 77. (C)Individual tumor volumes from each mouse following rechallenge with B16tumor cells (2×10⁵) on the right hind flank.

FIG. 15: Survival of C57BL/6 mice following treatment saline or CVA21 incombination with the control antibody or anti-CTLA-4. (A) Survival ofC57BL/6 mice following treatment with saline or CVA21 in combinationwith the control antibody or anti-CTLA-4. The end/termination of theexperiment was day 77. (B) Table showing median survival of mice fromeach treatment group.

FIG. 16: Tumor volumes following combination immunotherapy and CVA21virotherapy on B16-ICAM-1 murine melanoma tumors. (A) Average tumorvolumes (mm3)±S.E.M. on study days 27 from mice treated withsaline+control antibody, saline+anti-CTLA-4, saline+anti-PD-1,CVA21+control antibody, CVA21+anti-CTLA-4, CVA21+anti-PD-1 orCVA21+anti-CTLA-4+anti-PD-1. (B) Average tumor volumes (mm3)±S.E.M.following treatment with either saline+control antibody,saline+anti-CTLA-4, saline+anti-PD-1, CVA21+control antibody,CVA21+anti-CTLA-4, CVA21+anti-PD-1 or, CVA21+anti-CTLA-4+anti-PD-1. (C)Individual tumor volumes from each mouse following combinationimmunotherapy and CVA21 virotherapy on B16-ICAM-1 murine melanomatumors. C57BL/6 mice were injected with B16-ICAM-1 cells intradermallyon the hind flank. On days 7, 10, 13 and 16 mice were injectedintravenously with either saline or CVA21 (1×10⁸ TCID₅₀ [5.56×10⁹TCID₅₀/kg]), in combination with the control, anti-PD-1, anti-CTLA-4 oranti-CTLA-4+anti-PD-1 antibody (12.5 mg/kg respectively). Treatment daysare indicated by the dotted lines.

FIG. 17: Pt 12-002: Intra-tumoral CVA21 injection of metastatic melanomalesions on the leg of a male patient showing the effect on injected andnon-injected lesions at day 85. Pt 03-032: Male with metastatic melanomato left neck and lungs, showing non-injected distant visceral responseat day 86. Injection of CVA21 intratumorally in the left neck only.

FIG. 18: CALM Phase II trial. Preliminary analysis: Serum cytokineactivity (Patients with objective responses). Serum samples fromindividual patients were taken at the indicated times following theinitial intratumoral CVA21 injection. Serum samples were assessed forinflammatory cytokine levels as per manufactures instructions bymulti-plex immunoassay using a Bio-Plex Pro™ Human Cytokine 17-plexAssay (#M50-00031YV) BioRad, Richmond, Va., USA. Patient tumourresponses were assessed employing immune-related RECIST 1.1 criteria.

FIG. 19: Intravenous administered CVA21 (also noted on the figure asCAVATAK®) induces tumor cell gene expression changes. BALB mice wereadministered CVA21 or saline and sacrificed 3, 6, 24 and 72 hpost-administration and analysed for gene expression (right fourpanels). CVA21 replication kinetics are shown in the left single panel.

ABBREVIATIONS

Ab antibody

ACEC Animal Care and Ethics Committee

ANOVA Analysis of Variance

ATCC American Type Culture Collection

BSA bovine serum albumin

CI Combination Index

CO₂ carbon dioxide

CD cluster of differentiation

CPE cytopathic effect

CTL cytotoxic T lymphocyte

CVA21 Coxsackievirus A21

DAF decay-accelerating factor

dH₂O distilled water

DMEM Dulbecco's Modified Eagle's Medium

DNA deoxynucleotide phosphate

EtOH ethanol

FCS foetal calf serum

g gamma

ICAM-1 intercellular adhesion molecule-1

i.p. intraperitoneal

i.t. intratumoural

i.v. intravenous

irPFS immune-related progression-free survival

MAb monoclonal antibody

nAb neutralising antibody

MOI multiplicity of infection

PBS phosphate buffered saline

PCR polymerase chain reaction

PD-1 programmed cell death 1

PD-L1 programmed cell death 1 ligand 1

p.i. post inoculation

p.t.i. post tumour inoculation

RNA ribonucleic acid

RPMI Royal Park Memorial Institute

RT room temperature

RT-PCR reverse transcriptase-polymerase chain reaction

s.c. subcutaneous

SCID severe combined immunodeficient

SD standard deviation

SE standard error

sICAM-1 soluble intercellular adhesion molecule-1

TCID₅₀ Tissue Culture Infectious Dose 50%

UV ultraviolet

XTT2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide

Units

° C. degrees Celsius

d day

g gravitational force

g gram

h hour

L litre

m meter

M Molar

min minute

mol mole

rpm revolutions per minute

s second

U units

V volts

v/v volume per volume

w/v weight per volume

Nucleotides

A adenine

C cytosine

G guanine

T thymine

Prefixes

k kilo 10³

m milli 10⁻³

μ micro 10⁻⁶

n nano 10⁻⁹

p pico 10⁻¹²

DESCRIPTION OF EMBODIMENTS

The invention will now be described in more detail, including, by way ofillustration only, with respect to the examples which follow.

The following are some definitions that may be helpful in understandingthe description of the present invention. These are intended as generaldefinitions and should in no way limit the scope of the presentinvention to those terms alone, but are put forth for a betterunderstanding of the following description.

The methods of the invention typically involve administration of atherapeutically effective amount of the oncolytic virus or oncolyticviral RNA and of the immuno-stimulatory agent. The term “therapeuticallyeffective amount” as used herein, includes within its meaning anon-toxic but sufficient amount the oncolytic virus or oncolytic viralRNA and of the immuno-stimulatory agent for use in the invention toprovide the desired therapeutic effect. The exact amount required willvary from subject to subject depending on factors such as the speciesbeing treated, the age and general condition of the subject, theseverity of the condition being treated, the particular agent beingadministered and the mode of administration and so forth. Thus, it isnot possible to specify an exact “effective amount”. However, for anygiven case, an appropriate “effective amount” may be determined by oneof ordinary skill in the art using only routine experimentation.

In the context of this specification, the term “treatment” and relatedterms such as “treating”, “treated”, and treat” refer to any and alluses which remedy or alleviate a disease state or symptoms, prevent theestablishment of disease, or otherwise prevent, hinder, retard, orreverse the progression of disease or other undesirable symptoms in anyway whatsoever. For the avoidance of misunderstanding it is noted that“treatment” and related terms as used herein does not require completecure or remission of the disease being treated.

In the context of this specification, the term “comprising” means“including principally, but not necessarily solely”. Furthermore,variations of the word “comprising”, such as “comprise” and “comprises”,have correspondingly varied meanings.

To the extent permitted, the contents of documents referred to hereinare incorporated by reference.

In the context of this specification, the term “subject” or “patient”includes humans and individuals of any species of social, economic orresearch importance including but not limited to members of the genusovine, bovine, equine, porcine, feline, canine, primates, rodents. Inpreferred embodiments the subject or patient is a human.

Unless the context requires otherwise or specifically stated to thecontrary, integers, steps, or elements of the invention recited hereinas singular integers, steps or elements clearly encompass both singularand plural forms of the recited integers, steps or elements.

In the context of this specification, the singular encompasses also theplural except where the specific context clearly indicates otherwise.For example, where it is stated that the invention includes methods fortreatment of cancer by administration of an oncolytic virus or oncolyticviral RNA in combination with co-administration of an immuno-stimulatoryagent, it will be understood that this encompasses the administration ofone or more such viruses or viral RNAs and encompasses theadministration of one or more immuno-stimulatory agents.

In the context of this specification, the term “about” when used inrelation to a numerical value will be understood to convey the usualdegree of variation known in the art for the measure being described.Where the art does not recognise a usual degree of variation for ameasure or where it does and additional direction is neverthelessdesirable, the term “about” as used herein will be understood to conveya variation of plus or minus 10% of the numerical value to which theterm “about” is used.

Any description of prior art documents herein, or statements hereinderived from or based on those documents, is not an admission that thedocuments or derived statements are part of the common general knowledgeof the relevant art in Australia or elsewhere.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations or any two or more of said steps or features.

In the context of this specification, where a numerical range isprovided it will be understood to encompass the stated end points of therange and all values between those end points, including any sub-rangeswithin those endpoints.

The inventors herein demonstrate the use of immunostimulatory agentstargeting immune checkpoint molecules, exemplified herein by the use ofa murine anti-PD-1 antibody and a murine anti-CTLA-4 antibody, incombination with oncolytic CVA21 would be therapeutically beneficial inreducing tumour burden, rates of tumour ulceration and prolong survivalin a murine B16-ICAM-1 model of melanoma. Whilst it would be expectedthat enhancing the immune system by the use of immuno-stimulating agentswould disadvantage the efficacy of an oncolytic virus, for example byenhanced immune clearance or targeting of the virus, the inventorsherein demonstrate that surprisingly and counter-intuitively thecombination of oncolytic virus and immuno-stimulatory agent offersimproved anti-tumor effects compared to either agent virus orimmuno-stimulatory agent alone. The invention described herein thusprovides a method of treating tumours comprising delivering an oncolyticvirus or oncolytic viral RNA via direct injection or systemicadministration to the tumour in combination with the co-administrationof immuno-stimulatory agent via the systemic route to a mammal. Wherethe cancer desired to be treated is one for which a specific mechanismof delivery of virus or agent may be be advantageous, that method ofdelivery is preferred. For example, where the method is for thetreatment of bladder cancer or metastatic bladder cancer, administrationof at least the virus or viral RNA is advantageously by theintravesicular route.

CVA21 is a member of the human enterovirus C (HEC) family of viruses.Other notable members of the HEC family include the Coxsackieviruses,for example CVA13, CVA15, and CVA18. Each of CVA13, CVA15, CVA18 andCVA21 have been demonstrated to have oncolytic effect in the treatmentof various solid cancers, such as breast cancer, prostate cancer,colorectal cancer, ovarian cancer and melanoma (Shafren et al, 2004; Auet al., 2005; Au et al., 2007; WO2001/037866 filed 27 Nov. 2000 andentitled “A method of treating a malignancy in a subject and apharmaceutical composition for use in same”; the contents of which isincorporated herein in its entirety by reference) as well as hematologiccancers such as multiple myeloma (WO/2006/017914 filed 22 Aug. 2005 andentitled “Methods and compositions for treatment of hematologiccancers”, the contents of which is incorporated herein in its entiretyby reference). Each interacts with the ICAM-1 receptor for infection ofa host cell (Shafren et al, 1997) with decay accelerating factor (DAF)acting as a cooperative sequestration site (Shafren et al, 1997).Accordingly, the demonstration herein of a beneficial effect in a cancermodel of the co-administration of CVA21 with immune-stimulatory agentswill also apply to viruses functionally related to CVA21, such as CVA13,CVA15 and CVA18 and other human enterovirus C.

Any suitable source of the virus may be used in the methods of theinvention. For example, various suitable strains of virus may beobtained from the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209 USA, such as materialdeposited under the Budapest Treaty on the dates provided below, and isavailable according to the terms of the Budapest Treaty. Coxsackie groupA virus, strain CVA13 ATCC No.: PTA-8854 Deposited 20 Dec. 10 2007;Coxsackie group A virus, strain CVA15 (G9) ATCC No.: PTA-8616 Date ofDeposit: Aug. 15, 2007; Coxsackie group A virus, strain CVA18 ATCC No.:PTA-8853 Deposited 20 Dec. 2007; Coxsackie group A virus, strain CVA21(Kuykendall) ATCC No.: PTA-8852 Deposited 20 Dec. 2007.

As described herein the methods encompass the viral agent beingadministered as an oncolytic virus and the viral agent beingadministered in the form of oncolytic viral RNA, such as viral RNAcorresponding to CVA21. Methods for the use of oncolytic viral RNA inthe treatment of cancer have been described in WO2006/074526 entitled“Method and composition for treatment of neoplasms”, the contents ofwhich are incorporated herein by reference.

Following infection, an oncolytic virus can kill a cancerous cell bydirect lytic infection, induction of apoptosis or by initiating animmune response to viral antigens. An oncolytic virus is thus notlimited to a single input dose and can undergo a multi-cycle infection,resulting in the production of large numbers of progeny virus. Theseprogeny can spread either locally to adjacent tumour cells, orsystemically to distant metastatic sites. This feature of oncolytictherapy is particularly attractive for the treatment of inaccessibletumours or un-diagnosed micro-metastases. The demonstration herein, forexample, that intra-tumoral administration of CVA21 to a melanoma on theneck of a patient is associated with antitumour activity in bothinjected and distant non-injected lesions (FIG. 17), is consistent witha systemic effect occurring. As demonstrated herein intralesional CVA21is a promising novel oncolytic immunotherapeutic agent for the treatmentof unresectable Stage IIIC-IVM1c melanoma and the clinical studydescribed herein under Example 5 study met its primary endpoint of irPFSat 6 months. CVA21 was well tolerated and exhibited both local anddistant durable tumor responses.

The observation herein of antitumour activity in non-injected lesions inparticular is consistent with a systemic host immune-mediatedanti-tumour response. The Examples herein suggest that the CVA21mediated non-injected distant metatastic lesion activity was linked to ahost generated immune response as evidence by a possible novel serumcytokine signature of elevated levels of serum IL-8 and g-IFN (FIG. 18).Intravenous delivery of CVA21 in a mouse model, implanted in the flankwith melanoma cells (SK-Mel 28), was associated with an increase in theexpression of interferon-γ inducible protein 10 (IP-10) and PD-L1 by thetumor cells, as assessed by a timecourse of tumor biopsy followingadministration of the virus (FIG. 19). IP-10 is a chemokine secreted bycells exposed to IFN-g and plays an important role in recruitingactivated T cells into sites of tissue inflammation. In on-goingclinical studies it is becoming increasingly apparent that patientswhose tumours express higher levels of immune checkpoint molecules, suchas PD-L1 display superior tumour responses compared to patients whosetumour lack PD-L1 expression following treatment using anti-PD-1 oranti-PD-L1 blockade. This demonstrates the potential for CVA21 to act asa tumor immuno-agitator in combination with immune checkpoint inhibitorantibodies in ICAM-1 expressing cancers, such as melanoma, NSCLC,metastatic bladder cancer, kidney, multiple myeloma, pancreatic,glioblastoma and prostate cancers, and others.

The methods of the invention typically involve administration of atherapeutically effective amount of the virus and of theimmuno-stimulatory agent. The term “therapeutically effective amount” asused herein, includes within its meaning a non-toxic but sufficientamount of the virus or immuno-stimulatory agent, to provide the desiredtherapeutic effect. As noted herein, due to synergistic effects theamount of virus and immuno-stimulatory agent, used may be less than thatwhich would be used in a monotherapy (being treatment of a cancer in asubject using just one of the virus or the immuno-stimulatory agent).The exact amount required will vary from subject to subject depending onfactors such as the species being treated, the age and general conditionof the subject, the severity of the condition being treated, theparticular agent being administered and the mode of administration andso forth. Thus, it is not possible to specify an exact “effectiveamount” in the abstract. However, for any given case, an appropriate“effective amount” may be determined by one of ordinary skill in the artusing only routine experimentation.

The oncolytic virus or oncolytic viral RNA and the immuno-stimulatoryagent are used in conjunction with each other for, in one aspect, thetreatment of a subject having a tumour or having cancer. In embodimentswhere the method involves “co-administration” of the viral and theimmuno-stimulatory agents to a subject, it will be understood that thisterm means that the agents are administered so as to have overlappingtherapeutic activities, and not necessarily that the agents areadministered simultaneously to the subject. The agents may or may not bein physical combination prior to administration. Typically, the agentswill not be in physical combination prior to or when administered. In anembodiment the virus and the immuno-stimulatory agent(s) areadministered to a subject simultaneously or at about the same time. Inan embodiment the virus is administered to the subject before theimmuno-stimulatory agent(s) is administered.

The virus is typically administered to the subject in the form of apharmaceutical composition comprising virus and a pharmaceuticallyacceptable carrier. The composition may comprise the virus at anysuitable concentration, such as in a concentration range of about 10⁵viral particles per ml to about 10¹⁵ viral particles per ml, or about10⁶ viral particles per ml, or about 10⁷ viral particles per ml or about10⁸ viral particles per ml, or about 10⁹ viral particles per ml, orabout 10¹⁰ viral particles per ml, or about 10¹¹ viral particles per ml,or about 10¹² viral particles per ml, about 10¹³ viral particles per ml,or about 10¹⁴ viral particles per ml, or about 10¹⁵ viral particles perml.

A stock of the virus composition may be diluted to an appropriate volumesuitable for dosing, for example to achieve the desired dose of viralparticles administered in a desired volume. For example, a subject maybe administered a dose of virus comprising about 10⁵ viral particles toabout 10¹⁵ viral particles, or about 10⁶ viral particles, or about 10⁷viral particles, or about 10⁸ viral particles, or about 10⁹ viralparticles, or about 10¹⁰ viral particles, or about 10¹¹ viral particles,or about 10¹² viral particles, or about 10¹³ viral particles, or about10¹⁴ viral particles, or about 10¹⁵ viral particles. The volume in whichthe virus is administered will be influenced by the manner ofadministration. For example, administration of the virus by injectionwould typically be in a smaller volume, for example about 0.5 ml toabout 10 ml, compared to administration by intravesicular instillationin the case of treatment of bladder cancer, which may typically useabout 10 ml to about 100 ml, for example about 20 ml, about 30 ml, about40 ml, about 50 ml, about 60 ml, about 70 ml, about 80 ml or about 90ml, or in volumes similar to known procedures for instillation of BCGfor treatment of bladder cancer. As a further example, intravenousadministration of virus may typically use about 100 ml to about 500 mlof virus diluted in normal saline, infused by an automatic pump overapproximately 30 minutes.

Compositions may additionally include a pharmaceutically acceptablediluent, excipient and/or adjuvant. The carriers, diluents, excipientsand adjuvants must be “acceptable” in terms of being compatible with theother ingredients of the composition, and not unacceptably deleteriousto the recipient subject.

The virus may be administered as naked viral RNA encoding the virus,rather than viral particles, as described for example inPCT/AU2006/000051 entitled “Methods and composition for the treatment ofneoplasms”, filed 17 Jan. 2006, published as WO2006/074526, the entirecontents of which are incorporated herein by reference). In such anembodiment the viral RNA may be administered in the form of liposomes.Liposomes are generally derived from phospholipids or other lipidsubstances, and are formed by mono- or multi-lamellar hydrated liquidcrystals that are dispersed in an aqueous medium. Any non-toxic,physiologically acceptable and metabolisable lipid capable of formingliposomes can be used. The compositions in liposome form may containstabilisers, preservatives, excipients and the like. The preferredlipids are the phospholipids and the phosphatidyl cholines (lecithins),both natural and synthetic. Methods to form liposomes are known in theart, and in relation to this specific reference is made to: Prescott,Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y.(1976), p. 33 et seq., the contents of which is incorporated herein byreference.

The virus may be administered to the subject by any appropriate means,such as by injection. The injection may be systemically, parenterally,direct injection into the cancer, or intravesically. Typically, in thetreatment of bladder cancer the administration of the virus isintravesically (infused directly into the bladder).

Intralesional injection of a tumor may be performed by any appropriatemeans known to the skilled person, taking into account factors such asthe type of tumour being treated, the size and location of the tumor,accessibility of the tumour to direct injection. Injection techniqueswhich increase or maximise the distribution of the virus throughout thetumor may offer improved therapeutic outcomes. For example, in thetreatment of melanoma and other solid tumors, multiple lesions may beinjected in a dose hyper-fraction pattern, starting with the largestlesion(s) (2.0 mL injected into tumors >2.5 cm, 1.0 mL into 1.5 to 2.5cm; 0.5 mL into 0.5 to 1.5 cm) to a 4.0 mL maximum Following initialinjection with CVA21, any injected lesion that reduces in diameter to<0.5 cm may be injected with 0.1 mL of CVA21 as per the stated treatmentschedule until the lesion completely resolves.

Maximizing the number of cancer cells and regions throughout the tumorthat are initially infected theoretically will increase the amount ofcancer cells destroyed. It will also increase the amount of viralprogeny produced by the tumor, and therefore increase the chance ofongoing viremia for the seeding of remote tumors. Any appropriate meansto achieve desired distribution of the administered virus through thetumour may be used and will be apparent to the skilled addressee. Thefollowing describes one illustrative means, in the context ofadministration of CVA21 to a tumour, that the inventors have used inexperiments reported herein.

A syringe that will accommodate the volume of virus, such as CVA21,required and a 25-gauge needle are used for administration. The volumeof CVA21 to be administered may be determined based on the diameter ofthe tumor to be injected, as noted above. To load the syringe withCVA21, remove vial from individual carton and thaw at room temperature(18-25° C.). Do not leave the vial at RT for longer than is necessary tothaw the contents. Gently mix the vial for 5 seconds. Use a luer-locksyringe of appropriate volume and 21-gauge needle to draw up therequired volume. Remove air bubbles. Remove the withdrawal needle andreplace with a 25-gauge capped needle. Hold on ice until required (2-8°C.). Administer within 3 hours from loading the syringe as distributedinto the tumors as described below.

The injection may be in 9 regions within the tumor on each injectionday. The regions do not overlap, and they may be selected by using thefollowing landmarks. The distribution of the viral solution may be asfollows for Day 1, the first injection: (i) the center of the tumor isestimated and marked; (ii) marks are made around the periphery of thetumor at 45 degree radiants; (iii) the site of the needle insertion isbetween the center mark and the 270 degree radiant, at the approximatemidpoint—this is the first dose injection site; (iv) the volume ofdistribution is divided by 10, and will be distributed into 9 zoneswithin the tumor. The target zone for injection is the area within thetumor adjacent to the radiant marks, estimated to be approximatelywithin the outer 20% “rim” of the tumor. This will result in 8injections. These first 8 injections should be aimed to be deep to themidline plane of the tumor as guided by ultrasound. The final injectionis made directly deep to the predicted center of the tumor, and on thisfirst dose is aimed at a depth above the midline and comprises 20% ofthe injection volume.

It will be understood that the aforementioned calculation of amount ofvirus to be administered, as well as the described methods by which thevirus may be administered are provided only for the purpose ofillustration and are not intended to be limiting on the inventiondescribed herein.

The present invention provides methods for the treatment of cancer, themethods comprising the use of a human enterovirus C, such as CVA21, incombination with an immuno-stimulatory agent. The immuno-stimulatoryagents that have been the subject of much recent research and clinicaldevelopment are those that target the so-called checkpoint inhibitors.In checkpoint blockade humanised monoclonal antibodies are used tointerfere with host immune checkpoint molecules and their naturalligands. Blockade of such molecules including PD-1, PDL 1/L2 and CTLA-4has resulted in dramatic anti-tumor responses in large numbers ofadvanced cancer patients (melanoma, non-small cell lung cancer, bladderand renal cancers). Immune checkpoint molecules normally function tokeep the host immune system in balance and in maintainingself-tolerance. Specific blockade immune checkpoint molecules relaxesthe negative feedback system and elevates the activity of the hostimmune system to be more active, in particular to cancerous cells andantigens. In simple terms it takes the “biological handbrake” of thehost immune system. Immune checkpoint blockade has resulted in improveddurable tumor responses which have translated into meaning survivalbenefits. Despite the dramatic widespread tumor responses in largenumbers of cancer patients there remains a need to increase the rate anddurability of tumor responses elicited by immune checkpoint blockade.

As demonstrated herein, the combination of an oncolytic humanenterovirus C, such as CVA21, with an immune checkpoint blockademolecule (an immuno-stimulatory agent), such as an anti-PD-1 antibody oran anti-CTLA-4 antibody, provides surprising advantages in elicitingfavourable tumour response. Importantly, it has surprisingly beenidentified that administration of the virus induces changes to thestasis of the tumor microenvironment, with regard to impact onexpression levels of immune checkpoint molecules and host immune cellinfiltrates. As a number of these immune up-regulation processes involvethe direct activity of immune stimulating agents such as interferons, inparticular interferon-γ, therapeutic approaches involving administrationof human enterovirus C, such as CVA21, to induce intracellular viralreplication is an attractive process to initiate a targeted disruptionof the delicate balance of host immune system activities.

The immuno-stimulatory agent(s) may be selected from any appropriateagents. In the context of the invention an immuno-stimulatory agent willbe understood as any agent capable of stimulating an immune response totumor cells when administered to an individual. In preferred embodimentsof the invention the immuno-stimulatory agent may be any agent thatinteracts with an immune checkpoint molecule to block, diminish orcounteract the ability of that immune checkpoint molecule, or a complexcomprising that immune checkpoint molecule, in reducing the innateimmune-based anti-tumor responses of the individual. Hence, animmuno-stimulatory agent reduces the “handbrake” effect that the immunecheckpoint molecules have on the anti-tumor response. For example, theimmuno-stimulatory agent may be any agent that targets an immunecheckpoint molecule selected from the group consisting of PD-1, PD-L1,PD-L2, CTLA-4, CD134, CD134L, CD137, CD137L, CD80, CD86, B7-H3, B7-H4,B7RP1, ICOS, TIM3, GALS, CD28 or OX-40. It will also be understood thatthe term “immuno-stimulatory agent” as used herein may also be referredto as immune checkpoint inhibitors, when the immuno-stimulatory agenttargets an immune checkpoint.

Typically, the immuno-stimulatory agent may be an antibody, or inpreferred embodiments may be a monoclonal antibody. Preparation ofantibodies for use in the present invention may be carried out bymethods well known in the art, including preparing monoclonal antibodiesusing well known techniques and screening for high affinity antibodies,or by first identifying a monoclonal antibody having reasonably highaffinity and then improving the affinity using well known methods [e.g.,Huse, W. D., et al., Internat'l Rev. Immunol. 10: 129-137 (1993);Yelton, D. E., et al., J. Immunol. 155: 1994-2004 (1995); Wu, H., etal., Proc. Natl. Acad. Sci. (USA) 95: 6037-6042 (1998); Crameri, A., etal., Nature Medicine 2: 100-103 (1996); Stemmer, Proc. Natl. Acad. Sci.(USA) 91: 10747-10751 (1994); Stemmer, Nature 370: 389-391 (1994), theportion of each of which having to do with preparation of antibodies isincorporated herein by reference]. Alternatively, the agent may beobtained rather than prepared. Examples antibodies to checkpointinhibitor molecules include Nivolumab (BMS-936558, MDX-1106, ONO-4538),a fully human Immunoglobulin G4 (IgG4) monoclonal PD-1 antibody whichwas the first of its class to be tested in a phase I trial of 107patients with metastatic melanoma [Sosman et al. 2012b]. Lambrolizumab(MK-3475), a humanized monoclonal IgG4 PD-1 antibody, which was studiedin a phase I trial that included 132 patients with metastatic melanoma[Iannone et al. 2012]. BMS-936559, a fully human IgG4 PD-L1 antibody,was tested in 55 patients with metastatic melanoma as part of the phaseI trial [Brahmer et al. 2012].

In the treatment of a patient for a particular type of cancer theskilled addressee will be aware that additional methods or steps oftreatment may also be appropriate. For example, in the treatment of apatient for bladder cancer, the methods of the invention may optionallyinclude a bladder rinse or washout prior to administration of the virus,for example to prepare the bladder for improved receptivity of the virusby removing or reducing the presence of agents which may reduce theefficacy of the virus. For example, the urothelium is protected by aglycosaminoglycan (GAG) layer, disruption of which may permit moreefficient binding of the virus to cells and hence more efficienttransduction of cells. In a non-limiting example DDM(n-dodecyl-β-D-maltoside), a nonionic mild detergent used as a foodadditive and solublizing agent, may be used to disrupt or remove the GAGlayer at any appropriate concentration, for example at a concentrationof about 0.1%, and thereby assist in facilitating transduction.

The methods of the invention may be used in combination with surgicaltreatment of the cancer. For example tumor resection may be followed bytreatment of the subject using a combination method according to theinvention. It is anticipated that this may prevent or reduce recurrenceof the tumour.

The methods may comprise single or multiple doses of any one or more ofthe virus, or the immuno-stimulatory agent, such as an agent for examplea monoclonal antibody, that specifically binds to the surface expressedPD-1, PD-L1, PD-L2, CTLA-4 or OX-40.

The invention also relates to kits for use in the methods of theinvention. In a basic form, the kit may comprise a pharmaceuticalcomposition comprising the human enterovirus C and a pharmaceuticallyacceptable carrier, and instructions for the use of the composition, incombination with a chemotherapeutic agent or radiation, for thetreatment of cancer in a patient. The composition may be provided in anysuitable container, such as for example a vial, ampoule or syringe. Thecomposition may be provided lyophilised, freeze-dried, in liquid form orfrozen state.

The kit may comprise any number of additional components. By way ofnon-limiting example, additional components may include (i) one or moreanti-viral agents, such as Plecornil; (ii) one or more additionalpharmaceutical compositions comprising an oncolytic virus; (iii) one ormore additional pharmaceutical compositions comprising oncolytic viralRNA; (iv) one or more additional therapeutic agents useful in thetreatment of cancer in a patient. The kit may additionally comprise a animmuno-stimulatory agent for use in the combination therapy, such as amonoclonal antibody that specifically binds to the surface expressedPD-1, PD-L1, PD-L2, CTLA-4 or OX-40. The kit may also comprise of thecomposition being contained in a single-use vial, a pre-loaded syringefor direct human administration, diluted in a physiological solution forintravenous infusion or in a concentrated form enabling suitabledilution with physiological solutions. Such solutions may be, forexample, phosphate buffered saline or physiological concentrations ofNaCl₂.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of pharmaceutical compositions,such delivery systems include systems that allow for the storage,transport, or delivery of therapeutic agents (for example, oncolyticviruses in appropriate containers; or immuno-stimulatory agents inappropriate containers) and/or supporting materials (for example,buffers, written instructions for use of the compositions, etc.) fromone location to another. For example, kits include one or moreenclosures, such as boxes, containing the relevant components and/orsupporting materials.

The kit may be a fragmented kit. As used herein, the term “fragmentedkit” refers to a delivery system comprising two or more separatecontainers that each contain a subportion of the total kit components.The containers may be delivered to the intended recipient together orseparately. A fragmented kit may be suitable, for example, where one ormore components, such as the virus or the immuno-stimulatory agent, mayoptimally be stored and or transported under different conditions, suchas at a different temperature, compared to one or more other components.Indeed, any delivery system comprising two or more separate containersthat each contains a subportion of the total kit components are includedin the term “fragmented kit.” In contrast, a “combined kit” refers to adelivery system containing all of the components of a reaction assay ina single container (e.g., in a single box housing each of the desiredcomponents). The term “kit” includes both fragmented and combined kits.

EXAMPLES Example 1 Oncolytic CVA21 Virotherapy in Combination with theImmunostimulatory Antibody Anti-PD-1

This study investigated the effectiveness of CVA21 oncolytic virotherapyin combination with the immunostimulatory antibody anti-PD-1 in aB16-ICAM-1 murine model of malignant melanoma. Mice were implanted withtumours intradermally (2×10⁵ cells) and allowed to establish for 12 daysbefore commencement of therapy. Mice were treated with either saline,CVA21 or UV-inactivated CVA21, in combination with either a murineanti-PD-1 antibody or a matched control antibody on days 12, 15, 18 and21. The saline, CVA21 or UV-inactivated CVA21 treatments wereadministered intratumourally while the anti-PD-1 and control antibodieswere administered intraperitoneally. Animals treated with CVA21 incombination with the anti-PD-1 antibody showed a slight survival benefitcompared to animals in the control group (saline+control antibody) basedon the endpoint of tumour ulceration or a loss in bodyweight greaterthan 10%. During the treatment period between days 12 and 21, thecombination of CVA21+anti-PD-1 showed a tumoristatic effect, howeveronce therapy was stopped, the tumours slowly increased in volume.Despite this, the animals in this group showed a statisticallysignificant survival advantage compared to the saline+control antibody,UV-CVA21+control antibody and UV-CVA21+anti-PD-1 antibody treatmentgroups. This study also confirmed the immunostimulatory properties ofthe anti-PD-1 antibody, as anti-CVA21 neutralising antibody levels weredetected at higher levels in mice receiving CVA21+anti-PD-1 vsCVA21+control antibody. In summary the main finding of this study wasthat tumour bearing mice treated CVA21+anti-PD-1 showed an overallsurvival benefit that related to retardation of tumour growth andreduction in tumour ulceration.

Test Material: Virus

The test article, Coxsackievirus A21 (CVA21) [Trade name: CAVATAK™] wasprovided by Viralytics Ltd. Research stocks for in vitro use were madefrom a vial of commercially prepared CAVATAK™. Batch: CCVA2115;Concentration: 1×10⁹ TCID₅₀/ml (1.1 ml vials); Re-test Date:December-2008, January 2014 for Examples 2 and 3; StorageRecommendations: Store below −70° C.

The UV-inactivated CVA21 was prepared by exposing 7 ml of the sameCAVATAK™ Batch: CVA2115 product to UV-light for one hour in a biohazardhood. The virus was first transferred from the original product vials towells of a 6-well plate and exposed to the UV-light source at a distanceof approximately 10 cm for one hour. The UV-inactivated virus was thenaliquoted and frozen at −80° C.

Antibodies Control Antibodies

The antibody InVivoMAb Rat IgG2a from BioXCell was used as a controlantibody in Examples 1 and 2. Clone: 2A3. Catalog#: BE0089. Lot:4807/0713. Endotoxin: <0.35 EU/mg. Formulation: PBS, pH 6.5. Sterile:0.2 um filtration. Purity: >95%. Storage: 4° C. undiluted in the dark.The antibody InVivoMAb Rat IgG2b from BioXCell was used as a controlantibody in Example 3. Clone MPC-11. Catalog#BE0086. Lot 4700-2/0414.Endotoxin: <2.0 EU/mg. Formulation: PBS, pH 7. Sterile: 0.2 umfiltration. Purity: >95%. Storage: 4° C. undiluted in the dark.

Anti-PD-1

The active anti-murine PD-1 antibody was obtained from BioXCell. Productname: InVivoMAb anti m PD-1. Clone: RMP1-14. Catalog#: BE0416. Lot:4781/0813. Endotoxin: <0.61 EU/mg. Formulation: PBS, pH 7. Sterile: 0.2um filtration. Purity: >95%. Storage: 4° C. undiluted in the dark.

Anti-CTLA-4

The active anti-murine CTLA-4 antibody was obtained from BioXCell.Product name: InVivoMAb anti m CTLA-4. Clone: 9D9. Catalog#: BE0164.Lot: 5159/0414. Endotoxin: <2.0 EU/mg. Formulation: PBS, pH 7. Sterile:0.2 um filtration. Purity: >95%. Storage: 4° C. undiluted in the dark.

Cells

The human melanoma cell line SK-Mel-28 and murine melanoma cell linesB16 and B16-ICAM-1 were used in this study. The melanoma cell lineSK-Mel-28 was obtained from the ATCC (American Type Culture Collection).Mel-RM was a gift by Dr. P. Hersey (University of Newcastle, New SouthWales, Australia). B16 murine melanoma cells were originally obtainedfrom Dr A. Shurbier (Queensland Institute for Medical Research,Brisbane, Queensland, Australia), and then stably transfected with thehuman ICAM-1 gene to generate the cell line B16-ICAM-1. All cell lineswere maintained in DMEM (Thermo Scientific), containing 10% fetal calfserum (FCS) (SAFC Biosciences™, Australia), 10 mM sterileN-2-hydroxyethylpiperazine N′-2-ethanesulphonic acid (HEPES)(ThermoScientific, Australia), 2 mM L-glutamine (ThermoScientific,Australia), sodium pyruvate (Invitrogen, Australia) and 100 IU/mlpenicillin-streptomycin (Invitrogen, Auckland, NZ). All cells werecultured at 37 in a 5% CO₂ environment.

Animals

All animal work was approved by The University of Newcastle Animal Careand Ethics Committee (ACEC) under approval number: A-2013-327. Six toeight week-old female C57BL/6 mice (n=52) were obtained through theAnimal Services Unit of The University of Newcastle. They weremaintained in a specific pathogen-free area in the animal resourcesfacility within individually ventilated cages. Mice were housed ingroups of 4 within HEPA-filtered Techni-Plast Cages (1145 IVC) connectedto a Techni-Plast Slim Line air handling system within a PC2 laboratorywith a 12/12 hour light/dark cycle. Mice were fed ad libitum with mousecubes/pellets manufactured by Specialty Feeds, WA, Australia. Thisstandard mouse feed was formulated to be low in fat content(approximately 5%) and is meat free. The airflow in the room was 12 to15 air changes per hour but the airflow in the IVC cages was at a rateof 70 changes per hour. The mice were identified by tail markings with apermanent pen. All animal studies were conducted according to protocolsapproved by the Animal Ethics Committee. At the conclusion of the studyor when animals reached a humane endpoint mice were euthanased by CO₂asphyxiation.

Methods

Virus TCID₅₀ assay

Confluent monolayers of SK-Mel-28 cells in 96-well tissue culture plateswere inoculated with 10-fold serial dilutions (100 μL/well inquadruplicate) of CVA21 and incubated at 37° C. in a 5% CO₂ environmentfor 72 h. The mouse serum was serially diluted 10-fold ranging from1:10² to 1:10⁸ in DMEM containing 2% fetal calf serum (FCS). Wells werescored for cytopathic effects (CPE) visually under an invertedmicroscope. Wells that had detectable CPE were scored positive and the50% viral endpoint titre was calculated using the Karber method(Dougherty 1964).

Virus Neutralisation Assay

To test for the presence of neutralising antibodies, heat inactivatedmouse serum samples were first diluted in DMEM (2% FCS) 1:32 to 1:2048.One hundred microlitres of each serum dilution was incubated with 100 μLof CVA21 (100 TCID₅₀) at 37° C. for 1 hr. Fifty microlitres of thisserum/virus mixture was then plated in triplicate on SK-Mel-28 cells ina 384-well format. A +IgG (positive control) was obtained commerciallythrough Commonwealth Serum Laboratories (Sandoglobulin NF Liquid, Batchnumber: 4322800002). Wells were scored for the presence of virusneutralisation (lack of CPE) visually under an inverted microscope. Theneutralising antibody titre was then calculated using the Karber method(Dougherty 1964).

Immune-Competent B16-ICAM-1 Mouse Model of Melanoma

Fifty two female C57BL/6 mice (Animal Services Unit, The University ofNewcastle, Australia) aged between six to eight weeks were housed in thesame conditions as described above. The hind flanks of mice were shavedusing electric clippers three days prior to the injection of tumourcells to allow sufficient recovery time. B16-ICAM-1 cells were harvestedwith trypsin, washed twice and resuspended in sterile PBS. The viabilityof the prepared cells was assessed by trypan blue staining and analysiswith a TC-10 Automated Cell Counter (Biorad, Hercules, Calif., USA), andonly cell preparations with >95% viability were used forxenotransplantation. Prior to tumour transplantation, animals wereanesthetized with 5% isoflurane. Tumours were inoculated intradermallywith a single injection of 2×10⁵ B16-ICAM-1 cells in a volume of 50 titof PBS in the hind flank of mice.

The treatment groups and overview of the protocol used in Examples 1-3is briefly as follows.

In Example 1, animals were treated with either saline, CVA21(1×10⁸/injection) or UV-inactivated CVA21 (1×10⁸/injection), incombination with the control antibody or the anti-PD-1 antibody (12.5mg/kg respectively). Treatment was initiated 12 days post tumour cellimplantation with animals receiving the intratumoral treatment first(0.1 ml), followed by the intraperitoneal antibody (0.2 ml) immediatelyafter. Mice were given four courses of therapy every 3 days, starting atday 12 and ending on day 21 post tumour inoculation. A summary of thetreatment schedule used for Example 1 is shown in FIG. 1.

In Example 2 animals were treated with either saline or CVA21 (1×10⁸TCID₅₀/injection), in combination with either the control antibody orthe anti-PD-1 antibody (12.5 mg/kg respectively). Treatment wasinitiated 6 days post tumor cell implantation with animals receiving theintratumoral treatment first (0.1 ml/mouse), followed by theintraperitoneal antibody (0.2 ml/mouse) immediately after. Mice weregiven four courses of therapy every 3 days, starting at day 6 and endingon day 15 post tumor inoculation. Animals were given additional top-upinjections of saline of CVA21 (1×10⁸ TCID₅₀/injection) at weeklyintervals thereafter for a period of four weeks. A summary of thetreatment schedule used for Example 2 is shown in FIG. 8.

In Example 3 animals were treated with either saline, CVA21 (1×10⁸TCID₅₀/injection), in combination with the control antibody or theanti-CTLA-4 antibody (12.5 mg/kg respectively). Treatment was initiated7 days post tumor cell implantation with animals receiving theintratumoral treatment first (0.1 ml), followed by the intraperitonealantibody (0.2 ml) immediately after. Mice were given four treatmentsspaced 3 days apart, starting at day 10 and ending on day 16 post tumorinoculation. A summary of the treatment schedule used for Example 3 isshown in FIG. 12.

In Example 4 mice were treated with either intravenous saline orintravenous CVA21 (1×10⁸ TCID₅₀ [5.56×10⁹ TCID₅₀/kg assuming a 18 gmouse]), in combination with either a murine anti-PD-1 or anti-CTLA-4 oranti-PD-1+anti-CTLA-4 antibodies or a matched control antibody asdescribed above (12.5 mg/kg) on days 7, 10, 13 and 16. Saline or CVA21treatments were administered intravenously while the anti-CTLA-4, theanti-PD-1 and control antibodies were administered intraperitoneally(n=12-14 per group). As will be understood by the skilled addresseeintraperitoneal administration is an effective way of getting theantibody into the bloodstream and so provides an appropriate model forsystemic administration.

Tumours were measured twice a week and tumour volumes were estimatedbased on the volume of a spheroid V=Π/6·a·b² where “a” and “b” are thelongest and shortest perpendicular diameters of the tumour respectively.Blood from all mice were collected on a weekly basis by venipuncture ofthe saphenous vein and centrifuged at 10,000 rpm for 5 min at roomtemperature to collect the serum. Serum samples were stored at −80° C.until further testing. The animals were humanely euthanased by CO₂asphyxiation when tumours became ulcerated or loss in body weightexceeded 10%, otherwise mice were sacrificed at endpoint of study (day45 in Example 1; day 66 in Example 2; day 77 in Example 3).

TABLE 1 Immune-competent C57BL/6 mouse model: Mouse identificationnumbers and allocation of treatment groups Group 2 Group 3 Group 4 Group5 Group 6 Saline + Saline + UV CVA21 + UV CVA21 + Group 7 Control anti-Control CVA21 + Control CVA21 + Group 1 NTC Ab PD-1 Ab anti-PD-1 Abanti-PD-1 1515 1519 1523 1551 1539 1531 1559 1516 1520 1524 1552 15401532 1560 1517 1521 1525 1553 1541 1533 1561 1518 1522 1526 1554 15421534 1562 — 1527 1535 1555 1547 1543 1563 — 1528 1536 1556 1548 15441564 — 1529 1537 1557 1549 1545 1565 — 1530 1538 1558 1550 1546 1566

Statistical Analysis

All data was analyzed and plotted using GraphPad Prism v6.0 (GraphPadSoftware Inc.). For analysis of animal data, GraphPad Prism v6.0(GraphPad Software Inc.) was used to compare the difference in tumourvolumes between treatment groups using the two-way ANOVA (with repeatedmeasures) and Bonferroni post-tests. Comparison of survival curves wasperformed using the Log-rank (Mantel-Cox) test.

Results In Vivo Assessment of Combination Oncolytic Virotherapy andAnti-PD-1 Immunotherapy in an Immune-Competent Mouse Model

To assess whether the combination CVA21 and anti-PD-1 therapy approachwas effective in an immune-competent mouse model of melanoma, C57BL/6mice were implanted with murine B16-ICAM-1 tumour cells (2×10⁵)intradermally on the hind flank. Tumours were allowed to establishbefore commencement of therapy at 12, 15, 18 and 21 days post tumourinoculation (see FIG. 1). Saline or CVA21 was administeredintratumourally at the indicated time points, while the control antibodyor anti-PD-1 antibody were administered intraperitoneally. Mice weremonitored daily and weighed up to three times a week and tumour volumesmeasured by electronic calipers twice a week. Blood sampling from thesaphenous vein was carried out at weekly intervals. The study wasterminated at day 45 post tumour inoculation.

Body Weights Following Treatment with Either Saline, CVA21 orUV-Inactivated CVA21 in Combination with Anti-PD-1 or Control Antibody.

Animals were weighed up to three times a week and results recordedelectronically using FileMaker Pro and a proprietary animal monitoringdatabase for record keeping (Internal Ref: Experiment #53). Raw weightscan be found in Tables 2 to 10. Weights were also transcribed to theanimal monitoring checklist/records to meet the requirements of ourinstitutional Animal Care and Ethics Committee. No statisticallysignificant differences in the mean body weight were observed betweenthe treatment groups and NTC mice at any time points (multiple t-testscorrected for multiple comparisons using the Holm-Sidak method) exceptfor the UV-CVA21+anti-PD-1 group at day 31 (Prism 6 for Mac OS X Version6.0c, GraphPad Software, La Jolla Calif. USA, www.graphpad.com). Animalsappeared to tolerate the CVA21 and anti-PD-1 therapy well and there wereno observable toxicities from the treatments. A total of 9 animals wereeuthanased due to tumour ulceration and/or greater than 10% body weightloss. The decrease in weight was in the majority of cases linked totumour ulceration and associated tumour burden.

TABLE 2 Immune-competent animal model: Individual mouse body weights ofGroup 1 - No Tumour Control (NTC) mice. Group 1 - NTC - Mouse no. StudyDay 1515 1516 1517 1518 −2 15.34 16.13 18.02 17.23 0 15.6 16.86 17.6517.45 3 16.4 17.5 18.16 18.57 5 16.99 17.92 18.07 18.21 7 17.46 17.5418.31 18.2 10 17.41 18.45 18.25 18.22 12 17.97 18.11 19.29 18.47 1417.73 18.87 18.85 19.24 17 17.95 19.18 19.54 18.52 19 18.01 19.15 18.9619.3 21 18.94 19.15 19.4 20.18 24 18.52 19.02 19.32 19.43 26 19.81 20.0719.95 19.59 28 18.92 18.99 19.89 19.43 31 19.68 19.64 20.05 19.62 3319.03 19.45 20.2 20.22 35 20.21 20.12 20.06 20.85 38 19.89 19.96 20.2620.79 40 20 20.83 20.78 21.04 42 19.69 20.98 21.02 21.9 45 19.98 20.9520.68 22.32

TABLE 3 Immune-competent animal model: Individual mouse body weights ofGroup 2 - Saline + Control Ab treated mice. Group 2 - Saline + ControlAb - Mouse no. Study Day 1519 1520 1521 1522 1527 1528 1529 1530 −217.44 17.31 17.24 16.41 15.79 17.94 16.52 15.79 0 17.49 17.1 17.21 15.4615.74 17.11 17.13 15.75 3 17.59 17.6 16.96 16.39 15.73 17.77 16.31 15.855 17.71 17.61 16.85 16.02 16.13 18.14 17.08 16.41 7 18.37 18.11 17.7616.23 16.88 18.19 17.15 16.76 10 17.98 18.5 18.58 16.07 17.39 18.7217.46 17.29 12 19.2 19.14 18.63 17.01 17.36 18.62 17.54 16.9 14 — 18.4116.55 16.54 17.28 18.72 17.57 16.23 17 — 18.82 — 16.75 18.06 18.97 18.13— 19 — 19.58 — 17.32 17.86 19.08 17.86 — 21 — 19.31 — 17.4 17.22 19.5418.73 — 24 — 19.86 — 18.11 — 20.24 18.5 — 26 — 19.92 — 18.79 — 20.419.84 — 28 — 20.49 — 19.02 — 19.7 20.14 — 31 — 20.69 — 19.4 — — 21.43 —

TABLE 4 Immune-competent animal model: Individual mouse body weights ofGroup 3 - Saline + anti-PD-1 treated mice. Group 3 - Saline +anti-PD-1 - Mouse no. Study Day 1523 1524 1525 1526 1535 1536 1537 1538−2 16.03 17.41 16.25 16.95 17.18 15.65 15.25 17.14 0 16.66 17.63 16.0816.73 16.68 15.6 15.81 16.64 3 16.77 17.8 16.78 16.96 16.85 15.64 15.9316.88 5 17.61 18.56 17.07 17.53 17.07 16.29 16.21 17.18 7 18.08 18.3717.32 17.34 17.89 16.64 16.71 18.03 10 18.33 19.48 17.58 17.49 17.9216.75 17.53 18.12 12 18.57 19.17 18.35 16.83 18.41 16.72 17.69 18.87 1418.68 19.55 17.61 14.06 — 16.97 16.25 18.17 17 19.03 19.72 17.76 — — — —19.43 19 18.8 19.49 17.54 — — — — 19.95 21 19.47 20.38 17.93 — — — —20.22 24 18.96 20.14 18.08 — — — — 20.31 26 19.66 20.8 18.3 — — — —20.34 28 19.42 20.27 18.61 — — — — 20.6 31 20.37 21.3 18.24 — — — —20.65 33 — 20.9 18.52 — — — — 21.37 35 — 21.54 18.68 — — — — 21.58 38 —23.02 19.34 — — — — 22.4 40 — 23.47 19.91 — — — — 23.12 42 — — 20.34 — —— — — 45 — — 21.05 — — — — —

TABLE 5 Immune-competent animal model: Individual mouse body weights ofGroup 4 - UV CVA21 + Control Ab treated mice. Group 4 - UV CVA21 +Control Ab - Mouse no. Study Day 1551 1552 1553 1554 1555 1556 1557 1558−2 16.05 14.82 16.43 15.56 16.09 14.88 15.62 16.9 0 16.12 14.99 16.8815.42 15.97 15.23 15.79 16.64 3 16.66 15.45 17.32 16.07 15.92 15.6716.36 17.01 5 16.98 15.65 17.49 16.41 16.02 16.02 16.09 17.19 7 17.216.35 18.22 16.63 16.6 16.2 16.67 17.8 10 18.09 15.18 18.26 17.48 16.415.23 16.62 17.78 12 17.81 15.66 18.94 17.1 16.69 — — 18.2 14 17.9216.02 18.58 17.35 16.33 — — 17.74 17 — 16.03 18.66 17.64 16.62 — — 17.2719 — 16.12 18.77 17.05 17.13 — — 18.41 21 — 16.45 19.43 17.85 17.21 — —18.7 24 — 17.45 19.82 17.75 17.82 — — 19.41 26 — 17.71 21.26 18.66 17.9— — 19.89 28 — 17.91 18.66 18.7 19.14 — — 18.77 31 — 19.09 — 19.19 18.73— — — 32 — 17.9 — — — — — — 33 — — — 19.63 20.53 — — — 35 — — — — 20.88— — —

TABLE 6 Immune-competent animal model: Individual mouse body weights ofGroup 5 - UV CVA21 + anti-PD-1 treated mice. Group 5 - UV CVA21 +anti-PD-1 - Mouse no. Study Day 1539 1540 1541 1542 1547 1548 1549 1550−2 16.96 15.96 17.42 16.43 18.51 15.41 15.61 17.99 0 17.05 15.9 17.1516.45 17.39 15.82 15.34 17.34 3 16.96 16.5 16.97 17 18.72 16.8 15.918.09 5 17.53 17.06 17.38 17.14 18.18 16.88 15.51 17.87 7 17.88 17.0618.3 17.31 18.93 17.16 16.33 18.51 10 18.24 17.97 18.19 17.61 18.3717.95 16.38 18.17 12 18.93 17.71 19.14 17.72 19.84 18.23 16.47 18.58 1417.72 18.16 18.59 17.83 19.42 17.82 16.52 19.06 17 — 17.65 19.03 17.6819.57 18.48 16.93 18.78 19 — 18.11 18.73 18.36 19.94 19.21 16.87 19.2121 — 18.33 18.78 18.11 19.93 19.1 17.16 19.97 24 — 18.86 19.31 18.9520.38 20.08 17.46 20.27 26 — 19.49 20.44 18.62 21.02 19.42 18.15 21.5 28— 18.59 20.65 18.92 21.57 20.06 18.17 21.35 31 — 18.95 22.04 18.64 21.6619.93 — 22.79 32 — 19.29 21.37 — — — — — 33 — — — 18.81 23.07 20.78 —23.53 34 — — — — — 20.53 — — 35 — — — 18.94 23.62 — — — 38 — — — 19.78 —— — — 40 — — — 19.55 — — — — 42 — — — 20.03 — — — — 45 — — — 20.39 — — ——

TABLE 7 Immune-competent animal model: Individual mouse body weights ofGroup 6 - CVA21 + Control Antibody treated mice. Group 6 - CVA21 +Control Ab - Mouse no. Study Day 1531 1532 1533 1534 1543 1544 1545 1546−2 16.26 13.98 15.65 15.27 16.13 16.8 14.75 15.63 0 16.7 14.05 15.8715.48 16.34 16.19 14.59 15.77 3 16.75 13.69 16.06 15.81 16.53 16.8314.93 16.91 5 16.93 14.14 16.73 16.17 17.25 17.14 15.4 16.51 7 17.0615.05 17.19 16.67 18.09 17.2 15.7 17.46 10 16.85 14.75 17.32 16.81 18.2117.98 15.76 16.4 12 17.32 14.8 18.22 17.03 18.82 17.99 15.82 16.45 1416.88 14.69 17.19 16.91 18.47 17.52 16.13 — 17 17.04 15.25 18.59 17.1119.4 17.97 16.02 — 19 17.35 15.51 18.39 17.38 19.31 17.91 16.79 — 2117.69 15.64 18.65 17.44 19.69 18.19 16.41 — 24 17.84 14.33 18.96 18.1219.74 18.72 17.54 — 26 18.75 — — 17.57 21.15 19.61 17.64 — 28 18.77 — —— 19.91 20.33 18.27 — 31 18.51 — — — — 18.19 18.44 — 33 19.37 — — — — —18.98 — 35 19.97 — — — — — 20.35 — 38 20.96 — — — — — 21.01 — 40 21.68 —— — — — — — 42 22.37 — — — — — — — 45 24.67 — — — — — — —

TABLE 8 Immune-competent animal model: Individual mouse body weights ofGroup 7 - CVA21 + anti-PD-1 treated mice. Group 7 - CVA21 + anti-PD-1 -Mouse no. Study Day 1559 1560 1561 1562 1563 1564 1565 1566 −2 17.8816.47 16.52 19.57 15.37 16.5 16.68 17.03 0 18.02 16.59 16.73 19.24 16.4516.46 16.19 16.47 3 17.84 17.03 17.4 18.91 17.34 16.54 16.8 17.15 518.35 16.62 17.88 19.38 17.69 17.07 16.61 16.68 7 19.09 17.25 17.8919.98 17.82 16.77 17.17 17.28 10 19.63 17.6 18.53 20.61 19.05 17.5917.06 17.66 12 18.44 17.79 19.2 21.32 19.47 17.43 17.62 18.18 14 18.3917.45 19.16 20.23 19.12 17.37 17.57 17.88 17 19.34 17.24 18.92 21.0719.36 17.48 17.59 17.87 19 19.32 17.74 19.27 20.69 19.96 17.37 18.2518.11 21 19.8 17.54 19.54 21.25 20.54 17.38 17.58 18.13 24 20.02 18.2919.7 20.76 20.28 17.76 18.79 18.23 26 19.15 18.5 20.89 21.76 21 18.0118.69 18.61 27 18.25 — — — — — — — 28 — 18.84 19.82 21.67 21.68 18.6118.77 19 31 — 19.44 21.06 22.94 21.92 18.56 18.7 18.81 33 — 20.09 20.920.88 18.94 19.37 19.62 19.53 35 — 18.62 21.82 — — 19.09 19.8 19.94 36 —— — — — 16.21 — — 37 — 16.05 — — — — — — 38 — — 22.15 — — — 20.67 20.7740 — — 24.17 — — — 19.03 21.68 42 — — 24.15 — — — — 20.27 45 — — 23.23 —— — — 19.96

Tables 9, 10 and 11 are included in the Figures section of thisdocument.

Tumour Volume Data Following Treatment with Either Saline, CVA21 orUV-Inactivated CVA21 in Combination with Anti-PD-1 or Control Antibody.

Tumour volumes were measured twice a week using electronic verniercalipers. By day 31, all saline+control antibody treated mice wereeuthanased due to progressive disease evidenced by tumour volume tumourulceration. As shown in FIG. 4, little anti-tumour activity was observedin the UV-CVA21+control antibody treated tumours. By 35 days all tumourvolumes had escalated reaching the maximal humane endpoint and requiredeuthanasia. There was a high degree of variability in initial tumourstarting volumes and the most appropriate control for activeCVA21+anti-PD-1 was deemed the UV-CVA21+anti-PD-1 treated group ratherthan saline+anti-PD-1 (3 animals required euthanasia prior to completionof therapy). A comparison of tumour volumes at day 24 betweenUV-CVA21+anti-PD-1 and CVA21+anti-PD-1 treatments revealed that therewas a significant difference using a two-tailed t-test (p<0.0039). Thesignificant reduction in tumours between mice in the liveCVA21+anti-PD-1 group compared to the UV-inactivated CVA21 was mostnotable during the active phase of treatment and eventually the tumoursrelapsed in most groups.

Viral Clearance from Immune-Competent Mice and Increased Levels ofAnti-CVA21 Neutralising Antibodies

Infectious CVA21 was detected in the serum of virus treated animalsapproximately 45 minutes post intratumoural injection of virus on day12. The circulating virus in the blood was eliminated within the firstweek by day 19, despite additional treatments with CVA21intratumourally. Animals that were treated with live CVA21 produced thehighest levels of CVA21 neutralising antibodies, reaching a maximum atday 26. Given these mice have a functional immune system, the clearanceof CVA21 from the blood stream was not unexpected. Surprisingly, theadministration of the anti-PD-1 antibody appeared to enhance theanti-viral immune response, with these mice showing elevated levels ofanti-CVA21 antibodies of greater than 1:228 neutralising units, lastingup until day 45. This may be a result of the action of the anti-PD-1antibody allowing greater degree of viral replication and subsequentproduction of anti-viral antibodies. This finding raises the possibilitythat not only an enhanced level of anti-viral antibodies were producedbut a higher level of specific anti-tumour antibodies were generated inthis process, such anti-tumour antibodies may result in clinical benefitas a consequence of reducing tumour burden (antibody-dependent cellularcytotoxicity).

Enhanced Survival in Mice Treated with CVA21 in Combination withAnti-PD-1 Versus Saline and Anti-PD-1

CVA21 in combination with the anti-PD-1 antibody demonstrated astatistically significant improvement in overall survival compared tothe saline+Control antibody group (p=0.014 Log-rank [Mantel-Cox] test)(FIG. 7E). Comparing the saline+anti-PD-1 and saline+control antibodygroup, there was no statistically significant difference. This suggeststhat there was no difference between the control antibody and theanti-PD-1 antibody on survival. When the saline+anti-PD-1 survival curvewas compared with the CVA21+anti-PD-1 treatment group there was nostatistical difference (p=0.4901 Log-rank [Mantel-Cox] test) (FIG. 7B).This finding suggests that the main effect of survival was predominantlyanti-PD-1 based, however somewhat unexpectedly the comparison betweenUV-inactivated CVA21 and active CVA21 groups together with anti-PD-1revealed that live CVA21 gave an additional significant survival timeadvantage. Due to a high degree of starting tumour variability and earlyterm sacrifice, stringent analysis of relative survival benefits weresignificantly challenged. However a notable survival benefit trend oflive CVA21+anti-PD-1 treated group compared to the saline+anti-PD-1group was observed during the active treatment period.

Discussion

The results indicated that the use of live CVA21 in combination with theanti-PD-1 antibody gave the best overall survival compared to thesaline+control antibody treatment group. CVA21 in combination with theanti-PD-1 antibody demonstrated a statistically significant improvementin overall survival compared to the saline+control antibody group(p=0.014 Log-rank [Mantel-Cox] test) (FIG. 7E). Comparing thesaline+anti-PD-1 and saline+control antibody group, there was nostatistically significant difference. This suggests that there was nodifference between the control antibody and the anti-PD-1 antibody onsurvival. When the saline+anti-PD-1 survival curve was compared with theCVA21+anti-PD-1 treatment group there was no statistical difference(p=0.4901 Log-rank [Mantel-Cox] test) (FIG. 7B). This finding suggeststhat the main effect of survival was predominantly anti-PD-1 based,however addition of CVA21 did not enhance it significantly compared tosaline based on our limited group sizes. Interestingly the comparisonbetween UV-inactivated CVA21 and active CVA21 groups together withanti-PD-1 showed that live CVA21 gave an a slightly longer survival timeand was statistically significant. This fits our hypothesis thatactively replicating CVA21 and the lysis of tumour cells may stimulateanti-tumoural immunity more effectively in the presence ofimmunostimulatory anti-PD-1 antibodies compared to inactivated viralparticles. The survival of the UV-inactivated CVA21+anti-PD-1 antibodygroup was not statistically different to that of the UV-inactivatedCVA21+control antibody, or the CVA21+control antibody group.

An important finding of this study was that tumour bearing mice treatedCVA21+anti-PD-1 showed an overall survival benefit that related toretardation of tumour growth and reduction in tumour ulceration. Duringthe active phase of treatment, animals treated with the CVA21+anti-PD-1antibody showed a slowing of disease progression vs control groups.

Example 2 Oncolytic CVA21 Virotherapy in Combination with theImmunostimulatory Antibody Anti-PD-1: Tumor Rechallenge Study

This study was an extension of the work documented in Example 1,investigating the effectiveness of Coxsackievirus A21 (CVA21) oncolyticvirotherapy in combination with the immunostimulatory antibody anti-PD-1in a B16-ICAM-1 murine model of malignant melanoma. A timelinerepresentation of Example 2 is shown in FIG. 8. As different to Example1, the overall timeline of Example 2 was 66 days and included a “tumorre-challenge” as described in more detail below. Materials and methodsused in Example 2 were otherwise generally as described in Example 1,although the mice used for Example 2 were aged between four to sixweeks.

Mice were implanted with B16-ICAM-1 tumors intradermally (2×10⁵ cells)on the right hind flank and allowed to establish for 6 days beforecommencement of therapy. Mice were treated with either saline or CVA21(1×10⁸ TCID₅₀ [5.56×10⁹ TCID₅₀/kg]), in combination with either a murineanti-PD-1 antibody or a matched control antibody (12.5 mg/kg) on days 6,9, 12 and 15. Saline or CVA21 treatments were administeredintratumorally while the anti-PD-1 and control antibodies wereadministered intraperitoneally (n=12 per group). Additional top-upinjections of saline or CVA21 (1×10⁸ TCID₅₀ [5.56×10⁹ TCID₅₀/kg]) wereadministered at weekly intervals thereafter for a period of four weeks.The primary B16-ICAM-1 melanoma tumors were monitored regularly every 2to 3 days using digital calipers and tumor volumes calculated were basedon the formula for a spheroid using the two longest perpendicular axesin the x/y plane of the tumor. Animals with tumors showing signs ofulceration, weight loss of >10% or tumor volumes greater than 2500 mm³were euthanized.

At day 31, the remaining animals were intradermally rechallenged with1×10 B16 murine melanoma cells (that lacked the human-ICAM-1 receptor)to determine whether mice had developed a robust anti-tumoral immuneresponse following CVA21 virotherapy in combination anti-PD-1 therapy.Of the 37 animals that were re-challenged, all animals eventuallydeveloped palpable tumors, however there was a trend indicating that theonset of B16 tumor growth was delayed by CVA21+anti-PD-1 therapy ofexisting B16-ICAM-1 tumors.

Animals treated with CVA21 in combination with the anti-PD-1 antibodyshowed a statistically significant extension in survival compared toanimals in the saline+anti-PD-1 antibody and saline+control antibodygroup (median survival of 60 vs 45 vs 28 days respectively) suggestingthat CVA21+anti-PD-1 may be beneficial in a clinical setting. The use ofCVA21+anti-PD-1 was found to be well tolerated in this immunocompetentmouse model of melanoma with no adverse events relating to the agentstested.

Body Weights Following Treatment with Either Saline or CVA21 inCombination with Anti-PD-1 or Control Antibody

Body weights of individual animals were collected and analysed asdescribed in Example 1. No statistically significant differences in themean body weight were observed between the treatment groups and NTC miceat any time points (results not shown; multiple t-tests corrected formultiple comparisons using the Holm-Sidak method) [Prism 6 for Mac OS XVersion 6.0c, GraphPad Software, La Jolla Calif. USA, www.graphpad.com].Animals appeared to tolerate the CVA21 and anti-PD-1 therapy well andthere were no observable toxicities from the treatments. The decrease inweight was in the majority of cases linked to tumor ulceration andassociated tumor burden.

Tumor Volumes of Primary B16-ICAM-1 Nodules.

Tumor volumes were measured three times a week using electronic verniercalipers. By day 42, all saline+control antibody treated mice wereeuthanized due to progressive disease evidenced by tumor volume tumorulceration. As shown in FIG. 9, little anti-tumor activity was observedin the saline+control antibody treated tumors. All tumor volumes hadescalated reaching the maximal humane endpoint and required euthanasia.The CVA21+anti-PD-1 treatment group showed the best responses with anotable delay in tumor onset and only six animals showing signs ofprimary tumor growth towards the latter half of the study.

Tumor Volumes of Secondary B16 Nodules.

To establish whether a robust anti-tumoral immune response had developedfollowing CVA21 therapy in combination with anti-PD-1 treatment, micewere rechallenged with B16 murine melanoma cells. B16 cells lack thehuman ICAM-1 receptor and are therefore resistant to CVA21 therapy.These cells are antigenically similar to the B16 cells used to generatethe B16-ICAM-1 cell line and were used to identify the presence ofanti-tumoral immune responses that may have resulted following oncolysisof the primary tumor. As seen in FIG. 10, B16 tumors eventuallydeveloped in all of the mice rechallenged with B16 cells, however at day45 the average tumor volume of the saline+control antibody group wasstatistically larger than the saline+anti-PD-1 CVA21+control antibodyand CVA21+anti-PD-1 antibody groups (Two-way ANOVA).

Enhanced Survival in Mice Treated with CVA21 in Combination withAnti-PD-1 Versus Saline and Anti-PD-1.

CVA21 in combination with the anti-PD-1 antibody demonstrated astatistically significant improvement in overall survival compared tothe saline+Control antibody group (p<0.0001 Logrank [Mantel-Cox] test)(FIG. 11). Comparing the CVA21+control antibody and the saline+controlantibody group, there was no statistically significant difference. Whenthe saline+anti-PD-1 survival curve was compared with theCVA21+anti-PD-1 treatment group there was a statistical difference(p=0.0026 Log-rank [Mantel-Cox] test) (FIG. 11). This finding suggeststhat CVA21 used in combination with the anti-PD-1 antibody gave asignificant survival advantage (median survival of 45 vs 60 days forsaline+anti-PD-1 and CVA21+anti-PD-1 respectively).

Discussion

The Example 2 results indicated that the use of CVA21 in combinationwith the anti-PD-1 antibody improved the overall survival of tumorbearing mice compared to animals receiving the saline+anti-PD-1 antibodytreatment group. CVA21 in combination with the anti-PD-1 antibodydemonstrated a statistically significant improvement in overall survivalcompared to the saline+control antibody group (p<0.0001 Log-rank[Mantel-Cox] test) (FIG. 11). The treatment regime of CVA21 incombination with the anti-PD-1 was well tolerated with no adverse eventsattributed to the test articles. The main finding of this study was thattumor bearing mice treated CVA21+anti-PD-1 showed an overall survivalbenefit that related to retardation of tumor growth. Animals treatedwith the CVA21+anti-PD-1 antibody showed a slowing of diseaseprogression vs control groups and were more resistant to rechallengewith a secondary B16 tumor.

Example 3 Oncolytic CVA21 Virotherapy in Combination with theImmunostimulatory Antibody Anti-CTLA-4: Tumor Rechallenge Study

This study investigates the effectiveness of Coxsackievirus A21 (CVA21)oncolytic virotherapy in combination with the immunostimulatory antibodyanti-CTLA-4 in a B16-ICAM-1 murine model of malignant melanoma.

Mice were implanted with B16-ICAM-1 tumors intradermally (2×10⁵ cells)on the right hind flank and allowed to establish for seven days beforecommencement of therapy. Mice were treated with either saline or CVA21(1×10⁸ TCID₅₀ [5.56×10⁹ TCID₅₀/kg assuming a 18 g mouse]), incombination with either a murine anti-CTLA-4 antibody or a matchedcontrol antibody (12.5 mg/kg) on days 7, 10, 13 and 16. Saline or CVA21treatments were administered intratumorally while the anti-CTLA-4 andcontrol antibodies were administered intraperitoneally (n=12 per group).The primary B16-ICAM-1 melanoma tumors were monitored regularly every 2to 3 days using digital calipers and tumor volumes calculated based onthe formula for a spheroid using the two longest perpendicular axes inthe x/y plane of the tumor. Animals with tumors showing signs ofulceration, weight loss of >10% or tumor volumes greater than 2500 mm³were euthanased. CVA21+anti-CTLA-4 therapy in B16-ICAM-1 tumor bearingmice resulted in durable tumor regression compared to all othertreatment groups.

At day 37, the remaining animals were intradermally rechallenged with2×10⁵ B16 murine melanoma cells (that lacked the human-ICAM-1 receptor)to determine whether mice had developed a robust anti-tumoral immuneresponse following CVA21 virotherapy in combination with anti-CTLA-4therapy. Of the 34 animals that were rechallenged, all but six animalseventually developed palpable tumors (two saline+anti-CTLA-4 and fourCVA21+anti-CTLA-4 mice remained tumor free).

Animals treated with CVA21 in combination with the anti-CTLA-4 antibodyshowed a statistically significant extension in survival compared toanimals in the saline+control antibody group (median survival of 72 vs39 days) as did single agent CVA21 and anti-CTLA-4 antibody alone. Whilethere was no significant difference between CVA21+anti-CTLA-4 vs singleagent anti-CTLA-4, combination CVA21+anti-CTLA-4 gave improved survivalvs CVA21 alone (median survival of 72 vs 56.5 days) suggesting that thiscombination may be beneficial in a clinical setting. The use ofCVA21+anti-CTLA-4 was found to be well tolerated in this immunocompetentmouse model of melanoma with no adverse events relating to the agentstested.

Body weights following treatment with either saline or CVA21 incombination with anti-CTLA-4 or control antibody.

Body weights of individual animals were collected and analysed asdescribed in Example 1. No statistically significant differences in themean body weight were observed between the treatment groups and NTC miceat any time points (results not shown; multiple t-tests corrected formultiple comparisons using the Holm-Sidak method) [Prism 6 for Mac OS XVersion 6.0c, GraphPad Software, La Jolla Calif. USA, www.graphpad.com].Animals appeared to tolerate the CVA21 and anti-CTLA-4 therapy well andthere were no observable toxicities from the treatments. The decrease inweight was in the majority of cases linked to tumor ulceration andassociated tumor burden.

Tumor Volumes of Primary B16-ICAM-1 Nodules.

Tumor volumes were measured twice a week using electronic verniercalipers. By day 45, all saline+control antibody treated mice wereeuthanased due to progressive disease evidenced by tumor volume andtumor ulceration. As shown in FIG. 13, little anti-tumor activity wasobserved in the saline+control antibody treated tumors. All tumorvolumes had escalated reaching the maximal humane endpoint and requiredeuthanasia. The CVA21+anti-CTLA-4 treatment group showed the bestresponses with a notable delay in tumor onset and no animals showingsigns of primary tumor growth during the latter half of the study.Complete tumor regression followed by a durable response was observed in60% of animals treated with either monotherapies. More interestingly,all animals treated with CVA21+anti-CTLA-4 combination therapydemonstrated complete rejection against the primary tumor.

Tumor Volumes of Secondary B16 Nodules.

To establish whether a robust anti-tumoral immune response had developedfollowing CVA21 therapy in combination with anti-CTLA-4 treatment, micewere rechallenged with B16 murine melanoma cells. B16 cells lack thehuman ICAM-1 receptor and are therefore resistant to CVA21 therapy.These cells are antigenically similar to the B16 cells used to generatethe B16-ICAM-1 cell line and were used to identify the presence ofanti-tumoral immune responses that may have resulted following oncolysisof the primary tumor. At day 77, animals treated with theanti-CTLA-4+CVA21 combination therapy demonstrated 40% protectionagainst tumor rechallenge compared with 25% protection in the animalstreated with single agent anti-CTLA-4 antibody (FIG. 14).

Enhanced Survival in Mice Treated with CVA21 in Combination withAnti-CTLA-4 Versus Saline and Anti-CTLA-4.

Overall survival of animals on study. When compared against salinetreated animals, all treatment groups significantly extend the overallsurvival of the animals. Single agent anti-CTLA-4 (p=0.0068), singleagent CVA21 (p=0.0067) and CVA21+anti-CTLA-4+CVA21 (p=<0.0001). CVA21 incombination with the anti-CTLA-4 antibody demonstrated a statisticallysignificant improvement in overall survival compared to thesaline+Control antibody group (p<0.0001 Log-rank [Mantel-Cox] test)(FIG. 15). Comparing the CVA21+control antibody and the saline+controlantibody group, there was no statistically significant difference. Whenthe saline+anti-CTLA-4 survival curve was compared with theCVA21+anti-CTLA-4 treatment group there was a statistical difference(p=0.0026 Log-rank [Mantel-Cox] test) (FIG. 15). This finding suggeststhat CVA21 used in combination with the anti-CTLA-4 antibody gave asignificant survival advantage (median survival of 45 vs 60 days forsaline+anti-CTLA-4 and CVA21+anti-CTLA-4 respectively). Median survivalof animals on study is shown in FIG. 15(B). Animals receiving thecombination therapy of CVA21+anti-CTLA-4 had the longest median survival(72 days).

Discussion

The Example 3 results indicated that the use of CVA21 in combinationwith the anti-CTLA-4 antibody improved the overall survival of tumorbearing mice compared to animals receiving the CVA21+control antibodytreatment group. All CVA21+anti-CTLA-4 treated B16-ICAM-1 tumors hadregressed by day 77. CVA21 in combination with the anti-CTLA-4 antibodydemonstrated a statistically significant improvement in overall survivalcompared to the saline+control antibody group (p<0.0001 Log-rank[Mantel-Cox] test) (FIG. 15). By day 45 all saline+control antibodytreated mice were euthanased due to tumor progression and/or ulceration.The treatment regime of CVA21 in combination with the anti-CTLA-4 waswell tolerated with no obvious adverse events attributed to the testarticles.

The main finding of this study presented in Example 3 was that tumorbearing mice treated CVA21+anti-CTLA-4 showed an overall survivalbenefit that related to retardation of B16-ICAM-1 tumor growth. Animalstreated with the CVA21+anti-CTLA-4 antibody showed an inhibition ofdisease progression vs control groups and were more resistant torechallenge with a secondary B16 tumor compared to single agent treatedgroups.

Example 4 Intravenous Oncolytic CVA21 Virotherapy in Combination withthe Immunostimulatory Antibody Anti-PD-1, Anti-CTLA-4 and Anti-PD-1+AntiCTLA-4 Tumor Study

This study investigates the effectiveness of Coxsackievirus A21 (CVA21)oncolytic virotherapy in combination with the immunostimulatoryantibodies anti-PD-1, anti-CTLA-4 and anti-PD-1+anti CTLA-4 in aB16-ICAM-1 murine model of malignant melanoma.

Mice were implanted with B16-ICAM-1 tumors intradermally (2×10⁵ cells)on the right hind flank and allowed to establish for seven days beforecommencement of therapy. Mice were treated with either intravenoussaline or intravenous CVA21 (1×10⁸ TCID₅₀ [5.56×10⁹ TCID₅₀/kg assuming a18 g mouse]), in combination with either a murine anti-PD-1 oranti-CTLA-4 or anti-PD-1+anti-CTLA-4 antibodies or a matched controlantibody (12.5 mg/kg) on days 7, 10, 13 and 16. Saline or CVA21treatments were administered intravenously while the anti-CTLA-4 andcontrol antibodies were administered intraperitoneally (n=12-14 pergroup). The primary B16-ICAM-1 melanoma tumors were monitored regularlyevery 2 to 3 days using digital calipers and tumor volumes calculatedbased on the formula for a spheroid using the two longest perpendicularaxes in the x/y plane of the tumor. Animals with tumors showing signs ofulceration, weight loss of >10% or tumor volumes greater than 2500 mm³were euthanased.

Tumor Volumes of Primary B16-ICAM-1 Nodules.

Tumor volumes were measured twice a week using electronic verniercalipers. As shown in FIG. 16A, at study day 27 little anti-tumoractivity was observed in the saline+control antibody treated tumors. Allsingle agent groups (CVA21, anti-PD-1 and anti-CTLA-4) displayedsignificant tumor reductions compared to the saline group. Allcombination treatment groups exhibited tumor reductions compared to thesaline group but in general with greater significance levels withrespect to the single agent treatments. In FIG. 16B analysis of meantumor volumes throughout the entire study time course again indicatedsignificant tumor reductions in both single agent and combination groupscompared to the saline control animals. A notable trend was identifiedwith the combination of anti-PD-1 and CVA21 displayed reduced tumordevelopment kinetics compared to single agent CVA21 or anti-PD-1treatment alone. Individual spider plots of tumor development aredisplayed in FIG. 16C. The data indicate significant reduction in theincidence of papable tumor development in all treatment groups comparedto the saline control group. Of particular note is the reduction oftumor incidence in both anti-PD-1 and anti-CTLA-4 when combined withintravenous CVA21 administration compared to the presence of detectabletumors in the single agent anti-PD-1 or anti-CTLA-4 treated animals.More surprising, is the observation that all animals treated withCVA21+anti-PD-1+anti-CTLA-4 combination therapy demonstrated completerejection against the primary tumor development at study day 27.

Example 5 Systemic Effect of CVA21 Intra-Tumoral Injection on DistantTumours

Following intratumoural (i.t) injection, CVA21 preferentially infectsICAM-1 expressing tumour cells, resulting in tumour cell lysis and asystemic immune-mediated anti-tumour response. A Phase II trial of i.tdelivered CVA21 in advanced melanoma patients has highlighted antitumouractivity in both injected and distant non-injected lesions (FIG. 17).

The CALM study (CAVATAK™ in Late stage Melanoma) investigated theefficacy and safety of intratumoral administration of CVA21 in 57patients with treated or untreated unresectable Stage IIIC-IVM1cmelanoma. Each patient received CVA21 up to a total dose of 3×10^(8.0)TCID₅₀ (about 4.5×10⁶ TCID₅₀/kg for a 70 kg patient) in a maximum volumeof 4.0 mL by i.t. administration on Days 1, 3, 5, 8, 22, 43, 64 and atfurther 3-weekly intervals (up to a maximum of 10 sets of injections)until confirmed disease progression or development of excessivetoxicity. At each scheduled injection visit, if possible, multiplelesions were injected in a dose hyper-fraction pattern, starting withthe largest lesion(s) (2.0 mL of CVA21 injected into tumors >2.5 cm, 1.0mL of CVA21 into 1.5 to 2.5 cm; 0.5 mL of CVA21 into 0.5 to 1.5 cm) to a4.0 mL maximum, using ultrasound guidance if necessary. The length ofeach tumor to be injected is measured and the volume of CVA21 to beinjected into each tumor determined. The sum of these volumes is thetotal volume of CVA21 required for the administration. The maximumvolume of CVA21 to be administered is 4.0 mL Following initial injectionwith CVA21, any injected lesion that reduces in diameter to <0.5 cm wasinjected with 0.1 mL of CVA21 as per the stated treatment schedule untilthe lesion completely resolved. Patients displaying immune-relatedprogression-free survival (irPFS) or better at 6 months were eligiblefor 9 additional series of injections. Key eligibility criteria were ≧18yrs old, ECOG 0-1, and at least 1 injectable cutaneous, sc, or nodaltumor >1.0 cm The primary endpoint was to achieve >9 of 54 evaluablepatients with irPFS at 6 months following treatment; secondary endpointsincluded 1-year survival and objective response rates. A 2-stage Simon'sminimax design was employed. Thirty-five patients were treated in Stage1 with a futility clause requiring the observation of 3 or moreobjective responses (complete or partial; CR or PR, respectively)assessed by modified Response Evaluation Criteria in Solid Tumours(RECIST 1.1; Eisenhauer, et al., 2009) criteria in these patients toprogress to Stage 2. A further 22 patients were enrolled in Stage 2.

The primary endpoint of the study was achieved with 21 of 57 (38.6%)evaluable patients displaying irPFS at 6 months with a median irPFS of4.2 mos. The overall response rate (irRECIST) was 28.1% (16 of 57evaluable patients) with a ≧6 months durable response rate of 19.3% (11of 57 patients). The median time to response was 2.8 months, and the1-year survival rate 75.4% (43 of 57 patients). After a median follow-upof ˜16.5 months, median duration of response in responders and medianoverall survival (OS) for all patients was not reached. The most commonadverse events (AE's) were Grade 1 fatigue, chills, local injection sitereactions and fever. No Grade 3 or 4 product-related AE's were observed.

Antitumour activity in both injected and distant non-injected lesionswas evident in patients (FIG. 17), the latter observation in particularbeing consistent with a systemic immune-mediated anti-tumour response.CVA21 mediated non-injected distant metatastic lesion activity waslinked to a possible novel serum cytokine signature of elevated levelsof serum IL-8 and g-IFN indicating the generation of a potential activesystemic anti-tumor immune response.

Blockade of programmed death 1 (PD-1) and CTLA-4 in patients withmetastatic melanoma has resulted in substantial tumour responses via amechanism involving reversal of tumour induced T cell suppression. Asdemonstrated herein, the combination of CVA21 and PD-1 or CTLA-4blockade enhances antitumour responses, thereby offering improvedclinical activity.

Example 6 Intravenous Administered CVA21 Induces Tumor Cell GeneExpression Changes

Balb-C SCID mice were implanted on the left flank with SK Mel 28 cells(day 0). Mice were administered either CVA21 or saline intravenously byinjection into the tail vein (Day 14). Mice were sacrificed at 3 h, 6 h,24 h and 72 h post-treatment and the tumors excised for viral andcellular gene profiling. Upregulation of interferon-g inducible protein10 (IP-10) and PD-L1 was observed in tumour cells from mice treated withCVA21 (FIG. 19).

Cells, culture conditions, and virus were generally as described inExample 1. Female SCID-BALB/c mice of 6-8 weeks of age were obtainedfrom the ARC (Perth, Australia) and were housed under SPF conditionswithin the university animal holding facility. SK-Mel-28 cells weregrown in DMEM containing 10% FCS. Cells were harvested and washed twicein sterile PBS. Cell viability was assessed by trypan blue staining ascellular viability >95% was required for xenotransplantation. Cells wereresuspended in sterile PBS and kept on ice to maintain viability. Priorto xenotransplantation, mice were anaesthetised via isofluraneinhalation (4 L/min, maintained at 2%). Mice were given a sub-cutaneousinjection of 2×10⁶ SK-Mel-28 cells into the hind flank. Mice werevisually monitored daily and weighed every 3 to 4 days. Tumourdevelopment was monitored every 3 to 4 days by palpation. Tumours weremeasured using electronic callipers and estimates of tumour volume wereas described above (Example 1).

Once tumours were palpable (volume ≈50 mm³), mice were anaesthetisedwith isoflurane (4 L/min, maintained at 2%) and administered 1×10⁷TCID₅₀ CVA21 or sterile PBS (total volume 100 μL) via the retro-orbitalroute. Four mice from each tumour model, two of which had been treatedwith PBS and two of which had been treated with CVA21 were subsequentlysacrificed via CO₂ asphyxiation at 3, 6, 24 and 72 h post-treatment.Blood was taken via cardiac puncture. Tumours were excised and stored inRNALater (QIAGEN) for RNA stabilisation at 4° C. Serum (at a startingdilution of 1:10-1:100) was assayed for the presence of infectious virusvia the endpoint viral infectivity assay in triplicate, as follows.

To determine the titre of CVA21 in infected samples, SK-Mel-28 cellswere seeded in 96-well plates and grown to 50-80% confluency in DMEMcontaining 2% FCS. Cell monolayers were inoculated with 10-fold serialdilutions of purified CVA21 in triplicate or quadruplicate in DMEMcontaining 2% FCS and incubated at 37° C. in a 5% CO₂ environment for 72h. Wells that exhibited CPE upon microscopic examination were scored aspositive. Fifty percent infectious endpoint titres were calculated usingthe Karber method (Dougherty, 1964). Total RNA was extracted fromxenograft tissue using an RNEasy Mini Kit (QIAGEN) according to themanufacturer's protocol. Viral RNA was extracted from serum using theViral RNA Mini Kit (QIAGEN) according to the manufacturer's protocol.

RNA extracted from tumour and serum samples was analysed to determinethe levels of CVA21 RNA present using real-time quantitative RT-PCR. Onestep RT-PCR was carried out using the SuperScript III Platinum One-StepqRT-PCR Kit (Invitrogen). The primers and probe were specific for theVP3 region of the CVA21 (Kuykendall) genome and were designed usingPrimer Express 1.5 Software (Applied Biosystems, Foster City, Calif.).The sequence for the forward primer (KKVP3fwd) was5′-GAGCTAAACCACCAACCAATCG-3′ and the reverse primer (KKVP3rev) was5′-CGGTGCAACCATGGAACAA-3′. The FAM labelled probe (KKVP3) used was6FAMCACACACATCATCTGGGA-MGB. In a volume of 25 μL, the reaction mixturecomprised 1× SuperScript reaction mix, 500 nM forward primer, 500 nMreverse primer, 250 nM probe, 500 nM ROX, 0.5 μL SuperScript IIIRT/Platinum Taq Mix and 5 μL extracted RNA. RT-PCR reactions werecarried out using the ABI Prism 7000 Sequence Detection System (AppliedBiosystems). Cycling conditions were 30 min at 60° C., followed by 5 minat 95° C. and then 50 cycles of 15 sec at 95° C. and 1 min at 60° C.Samples were quantitated against pre-validated CVA21 RNA standards ofknown concentration and results reported as equivalent TCID₅₀/mL forserum RNA or TCID₅₀/mg for xenograft tissue RNA.

cRNA was amplified and a biotin-dUTP label incorporated. BiotinylatedcRNA samples obtained from SK-Mel-28 xenografts were hybridised toHumanRef-8 v2 Expression BeadChips (Illumina), representing >22 000transcripts, according to the manufacturer's protocol. Beadchip arrayswere scanned using the BeadStation 500 System (Illumina). Microarraydata were analysed using GeneSpring 7.0 software (Silicon Genetics,USA). Data sets were transformed by setting measurements <0.01 to 0.01and then further normalised per chip to a 50th percentile and per geneto a median. Within each xenograft model, data sets from each pair ofmice obtained for each treatment (PBS and CVA21) and time point (3, 6,24 and 72 h) were analysed as replicate-samples. Genes were filteredbased on positive gene expression (signal intensity) in at least one ofthe replicate-samples.

Intracellular viral replication is an attractive process to initiate atargeted disruption of the delicate balance of host immune systemactivities. In FIG. 19, systemic delivery of CVA21 to a human melanomaxenograft in a mouse model induced significant targeted viralreplication as evidence by the increasing levels of CVA21 specific RNAwithin the tumor tissue throughout the duration of the study, startingin particular at 24 h post-systemic administration via the retro-orbitalroute. Gene expression analysis at 3, 6, 24 and 72 hrs post-CVA21revealed significant up-regulation of interferon responses gene withinthe tumor microenvironment, in particular the interferon inducibleprotein-10 (IP-10), a chemokine secreted from cells exposed to IFN-g andwhich plays an important role in recruiting activated T-cells into sitesof tissue inflammation. As IFN-g provided by activated T-cells is knownto up-regulate immune checkpoint molecules, expression levels of thePD-L1 gene were monitored. As illustrated in FIG. 19, as CVA21replication peaked at 24-72 hrs post systemic administration, inparallel an accompanying increase IP-10 and PD-L1 expression, indicatingIFN-g activity. Based on the immune agitation induced by targeted CVA21replication in the melanoma tumor tissue, the inventors anticipate thatthis immune stimulating event may increase the anti-tumor activity ofimmune-checkpoint blockade when both agents are used in combination.

REFERENCES

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1. A method for the treatment of cancer in a subject, the methodcomprising delivering an oncolytic virus or oncolytic viral RNA viadirect injection to a tumor or systemic administration to the subject incombination with the co-administration of an immuno-stimulatory agentvia the systemic route to the subject.
 2. A method for the treatment ofbladder cancer in a subject, the method comprising delivering anoncolytic virus or oncolytic viral RNA via direct injection to a tumoror systemic administration or intravesicular administration to thesubject in combination with the co-administration of animmuno-stimulatory agent via the systemic route to the subject.
 3. Themethod of claim 1 or 2, wherein the oncolytic virus or oncolytic viralRNA is selected from the group consisting of Family Picomaviridae. 4.The method of claim 1 or 2, wherein the oncolytic virus or oncolyticviral RNA selected from the group consisting of Family Picomaviridaevirus that bind to intercellular adhesion molecule-1 (ICAM-1) and/ordecay-accelerating factor (DAF) on the surface of the tumour cell. 5.The method of claim 1 or 2, wherein the oncolytic virus or oncolyticviral RNA is selected from the group consisting of genus enterovirusthat bind to intercellular adhesion molecule-1 (ICAM-1) and/ordecay-accelerating factor (DAF) on the surface of the tumour cell. 6.The method of claim 1 or 2, wherein the oncolytic virus or oncolyticviral RNA is selected from the group consisting of Group ACoxsackievirus that bind to intercellular adhesion molecule-1 (ICAM-1)and/or decay-accelerating factor (DAF) on the surface of the tumourcell.
 7. The method of claim 1 or 2, wherein the oncolytic virus oroncolytic viral RNA is Coxsackievirus A21.
 8. The method of claim 1 or2, wherein the immunostimulatory agent is selected from the groupconsisting of a agent that specifically binds to the surface expressedPD-1, PD-L1, PD-L2, CTLA-4 or OX-40.
 9. The method of claim 1 or 2,wherein the immunostimulatory agent is selected from the groupconsisting of a monoclonal antibody that specifically binds to thesurface expressed PD-1, PD-L1, PD-L2, CTLA-4 or OX-40.
 10. The method ofclaim 1 or 2, wherein delivering the oncolytic virus or oncolytic viralRNA via direct injection or systemic administration or intravesicularadministration is prior to the administration of an immuno-stimulatoryagent via the systemic route.
 11. The method of claim 1 or 2, whereindelivering the oncolytic virus or oncolytic viral RNA via directinjection or systemic administration or intravesicular administration isfollowing the administration of an immuno-stimulatory agent via thesystemic route to the subject.
 12. The method of claim 1 or 2, whereinthe cancer or tumour is selected from the group consisting of prostatecancer, breast cancer, ovarian cancer, lymphoid cancer, leukemia, braincancer, lung cancer, colorectal cancer, thyroid cancer, renal cancer,adrenal cancer, liver cancer, stomach cancer, intestinal cancer, bladdercancer, cancer of the kidney, multiple myeloma, non-small cell lungcancer (NSCLC), pancreatic cancer, glioblastoma and melanoma.
 13. Themethod of claim 1 or 2, wherein the cancer or tumour is melanoma. 14.Use of oncolytic virus or oncolytic viral RNA for the manufacture of amedicament for the treatment of a subject having cancer, wherein saidmedicament is for use in combination with an immuno-stimulatory agentdelivered via the systemic route to the subject, and wherein saidmedicament is for delivery via direct injection to the tumor or systemicadministration to the subject.
 15. Use of oncolytic virus or oncolyticviral RNA for the manufacture of a medicament for the treatment of asubject having bladder cancer, wherein said medicament is for use incombination with an immuno-stimulatory agent delivered via the systemicroute to the subject, and wherein said medicament is for delivery viadirect injection to a tumor or systemic administration or intravesicularadministration to the subject.
 16. The use according to claim 14 or 15,wherein the oncolytic virus or oncolytic viral RNA is for administrationto the subject via direct injection to a tumour or systemicadministration or intravesicular administration to the subject prior tothe administration of an immuno-stimulatory agent via the systemic routeto the mammal.
 17. The use according to claim 14 or 15, wherein themedicament comprising an oncolytic virus or oncolytic viral RNA is foradministration to the subject via direct injection to a tumor orsystemic administration or intravesicular administration to the subjectfollowing the administration of an immuno-stimulatory agent via thesystemic route to the subject.
 18. An oncolytic virus or oncolytic viralRNA for use in treatment of a subject having cancer, wherein said use isin combination with an immuno-stimulatory agent, wherein in said use theoncolytic virus or oncolytic viral RNA is administered to said subjectvia direct injection to a tumor or systemic administration to thesubject and said immuno-stimulatory agent is administered via thesystemic route to the subject.
 19. An oncolytic virus or oncolytic viralRNA for use in treatment of a subject having bladder cancer, whereinsaid use is in combination with an immuno-stimulatory agent, wherein insaid use the oncolytic virus or oncolytic viral RNA is administered tosaid subject via direct injection to a tumor or systemic administrationor intravesicular administration to the subject and saidimmuno-stimulatory agent is administered via the systemic route to thesubject.
 20. The oncolytic virus or oncolytic viral RNA for useaccording to claim 18 or 19, wherein the administration of saidoncolytic virus or oncolytic viral RNA is prior to the administration ofthe immuno-stimulatory agent.
 21. The oncolytic virus or oncolytic viralRNA for use according to claim 18 or 19, wherein the administration ofsaid oncolytic virus or oncolytic viral RNA is after the administrationof the immuno-stimulatory agent.
 22. The method according to claim 1 or2, wherein the subject is a human.